Probing Proteins

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By Deborah Komlos

Unravelled genomes in hand, researchers worldwide are charting an ambitiously concentrated yet broad course. They aim to elucidate the many protein products of gene expression, not only identifying what they are and how they look, but also eyeing the larger picture by striving to reveal and understand the gamut of cell functions they perform. Canada continues to play a prominent role in this proteomics puzzle.

Among the Canadian projects underway are several proteomics endeavours based in Ontario and Quebec that have received Genome Canada (Ottawa, ON) funding through their respective Genome Centres, the Ontario Genomics Institute and Genome Quebec. The main funding and information resource relating to genomics and proteomics in Canada, Genome Canada recently held two funding competitions for large-scale genomics research projects and science and technology platforms that will facilitate current or newly proposed projects.

Those granted funding are what Anie Perrault, vice-president of Communications with Genome Canada, describes as “high-quality projects that will make Canada a world leader.”

A multidisciplinary panel of international experts assesses the funding applications, Perrault explains, and awards support to those meeting specific scientific, financial and management criteria such as scientific excellence of the proposed research, effectiveness of financial planning and quality of the management plan, respectively. Based on a project’s three-year budget, financial support from Genome Canada covers 50 per cent of eligible costs and requires the applicant(s) to supply the remainder from other sources.

Protein Groundwork

One of the funded platforms is the $8-million Proteomics Technology Core Facility led by Jack Greenblatt, PhD with the University of Toronto (Toronto, ON).

The facility was established to support proteomics research with one goal being to survey the proteins in a cell. “Frequently in a disease state, you have an alteration of the protein composition of a cell,” Greenblatt says. “If you can learn how protein composition of a cell relates to specific disease states then you have a very good way of diagnosing disease.”

To this end, two hospitals and seven universities in Ontario are involved in one “core” of the facility — the general characterization of proteins — to develop better mass spectroscopy methodology for protein analysis and identification.

Another defined “core” has structural proteomics at its heart. Led by researchers Cheryl Arrowsmith, PhD and Aled Edwards, PhD, both with the University of Toronto, the team is developing technologies for determining protein structures with particular emphasis on membrane-associated proteins, which Greenblatt says are the most frequent drug targets, and the ones for which it is the most difficult to determine structure.

The final “core” focus is to survey as many proteins as possible in individual organelles, work being led by Don Mahuran, PhD with the Hospital for Sick Children (Toronto, ON).

Currently, Greenblatt says the Proteomics Technology Core Facility’s University of Toronto location is being occupied entirely by the bacterial and yeast identification efforts of another Genome Canada-funded project, the $27-million Functional Genomics and Proteomics of Model Organisms, of which $11 million is allocated to proteomics.

Involving Greenblatt, along with project leader Janet Rossant, PhD and Anthony Pawson, PhD, both with the Samuel Lunenfeld Research Institute (Toronto, ON), and Andrew Spence, PhD and Brenda Andrews, PhD, both with the University of Toronto, the project aims to identify and characterize protein complexes and protein interactions in bacteria, yeast, C. elegans, mouse and mammalian cells.

Greenblatt is heading one of Andrews’s yeast subprojects and is also in charge of the bacteria component, which is focused on E. coli. The task is grand, involving the purification of each of the approximately 6,000 yeast proteins and 4,000 E. coli proteins. So far, Greenblatt says the team has done nearly half of the yeast proteins and about 700 of those in E. coli.

“Each time you purify a protein, with other proteins that are associated with it, you create an entire web of protein interactions for these cells,” Greenblatt says.

It is specifically an interaction web that Andrews and her lab are eager to build through their “molecular blueprint” for yeast.

The project goal, Andrews explains, is to generate a comprehensive understanding of the biology of a model eukaryotic cell. One approach uses proteomics to try to view the protein complexes in budding yeast (the work being headed by Greenblatt), and the other involves functional genomics through two methods: DNA expression profiling using microarrays, and a novel technology developed by Andrews and colleagues called synthetic genetic array (SGA) analysis.

“The technology (SGA) exploits the fact that a consortium of academic researchers has made a set of yeast strains in which each of the non-essential genes has been deleted and replaced by an antibiotic-resistance marker,” Andrews says.

Using that set, Andrews and her team have created an array of approximately 5,000 double mutants that is then scored for synthetic death, whereby the combination of two mutations causes cell death, or synthetic growth defects.

“The reason that’s important is because it’s clear — since you can delete 5,000 of the 6,000 genes in yeast and the cells still survive — that there are a lot of problems with genetic redundancy, and if that’s a problem for yeast, it’s clearly going to be a problem for higher eukaryotic cells,” Andrews explains.

“We want to be able to use a phenotypic read-out to understand gene function,” she adds. “So we’re comprehensively making double mutants and the idea is that we can use that information and build a genetic interaction network, which is actually proving to be highly informative about gene function.”

Role Integration

Elucidating function in proteomics research is a key component in several of the research endeavours, including the project headed by Rafick-Pierre Sékaly, PhD with the University of Montreal and David Kelvin, PhD at the Toronto General Research Institute (Toronto, ON), titled Functional Genomics, Pharmacogenomics and Proteomics of the Immune Response in Health and Immune Related Disorders.

Sékaly says the goal of the $15-million proposal (of which about $4 million is allocated to proteomics) is to generate a model of the immune response based on aberrant immune reactions that the team is observing in models demonstrating a very deficient immune system. Examples he gives include chronic viral diseases and conditions in which the immune system is hyperactive, such as transplantation or rheumatoid arthritis auto-immune diseases — diseases that cause the immune system to destroy the host rather than fight external pathogens.

“One problem of genomics is that when you’re looking at RNA, you’re looking at RNA from many different cells at the same time. And the immune system is very heterogeneous, so you can look at the whole thing. But I think it is also important to look at the contribution of every part of the immune response individually, so then you can integrate everything,” Sékaly explains.

With this in mind, the project will involve creating a model to analyse the immune response using existing and optimizing tools, such as the Fish and Chips assay that permits viewing the transcription profiles of 11 different genes on the same cell, and developing new technologies.

Current methods, he continues, may give a global picture by allowing the viewing and analysis of numerous cells simultaneously. But with Fish and Chips, “you know which cell from the whole mixture is giving you that response,” he says.

While Sékaly’s focus in Montreal is on functional assays, using cell sorters to generate the cell subsets, Kelvin’s facility works on the gene array work, an arrangement that Sékaly praises as it permits the establishment of standards by maintaining each operation at one site or the other.

For Sékaly, deciding to concentrate on the immune response was a logical extension of the work he is already involved in with CANVAC (Canadian Network for Vaccines and Immunotherapeutics) (Montreal, QC), for which he is the scientific director and program leader. “Rather than stay in the context of our more limited assays, we move now to the new generation of genomic and proteomic assays,” he says.

Through understanding the genes involved in regulating the immune system, the team hopes to be able to explain why individuals react differently to immune-based treatments, something that Sékaly says can have future application in simple tools to predict immune response to drugs.

He mentions the SARS epidemic in which there have been deaths as well as cases of convalescence, demonstrating variability in immunity. “Some people are able to combat and control the disease, while other people don’t have that capacity,” he explains. Similarly, some people respond well to vaccination while for others, the vaccine confers minimal or no protection. “It’s something that we need to understand,” he says.

Branching Out

A $13-million project led by John Bergeron, PhD with McGill University (Montreal, QC), Montreal Network for Pharmaco-Proteomics and Structural Genomics is a highly integrative, collaborative network involving 13 principal investigators from labs in several institutions around the city of Montreal. The network’s goal: to characterize all proteins in mammalian cells. For promotional and communications purposes, the granted project name has been simplified to Réseau Protéomique de Montréal, Montreal Proteomics Network (RPMPN).

Three main research projects make up the network, with the first to develop a comprehensive mammalian cell map which includes characterizing proteins, post-translational modifications and association in complexes in all cell compartments. A second, to map and validate all protein-protein interactions and post-translational modifications in eukaryotic organisms in normal and disease states. Structural genomics is the third and will involve nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography to determine structures for proteins identified from the cell map.

Moving in at summer’s end, the RPMPN will be housed at the McGill University and Genome Quebec Innovation Centre (Montreal, QC). Program manager for the RPMPN, Sean Taylor, PhD sees the core facility at McGill as a virtual lab, consisting of many peripheral groups working physically in their own teams but interlinked through the bioinformatics team.

The team developed a database system called CellMapBase through which the investigators have access to data generated from all of the research teams.

“For example, if you find a really interesting protein out of your work you might want to know is that protein actually located in other organelles, and you can determine that very quickly through CellMapBase,” Taylor says. “We’re all working not only for the goals of the individual labs, but also for the goals of the entire network. It’s a very functional team and there’s a real vision there, a real collaborative atmosphere.”

He strongly attributes the network’s success to its administrative group, including the recent hiring of Benoit Houle, PhD as scientific manager, to oversee the interlinkages between the scientists and their projects and track their work.

“How do you even decide on how a project can be defined using a proteomics approach, because proteomics goes on forever,” Taylor says. For instance, he adds, a group may determine a baseline level of proteins in a human cell, but a multitude of conditions, such as drug-induction or heat-shock, can cause the proteome to change.

“So even defining projects becomes a challenge and that’s something that we’re faced with every day in the proteomics network where we have to carefully plan and decide with the individual team members, what the beginning, middle and end of a project is,” he says.

Collaborations can also cross borders, as is the case with the $95-million partnership, the Structural Genomics Consortium (SGC). Led by Aled Edwards, PhD, the SGC is an initiative involving the University of Toronto and Oxford University (Oxford, UK), funded by the U.K.-based research charity The Wellcome Trust, GlaxoSmithKline (London, UK) and a few Canadian funding organizations, including Genome Canada, which contributed $15 million.

The consortium aims to determine the structure of over 350 human proteins, all of which will be immediately released to the public domain once solved. Edwards says the group is trying hard to meet its milestone of releasing the first structure by the year’s end.

Decisions on what specific diseases or disorders the group’s work may target have not yet been made, a situation that Edwards says is not necessarily significant early on.

“Many of these proteins have applications in lots of diseases,” Edwards says. “Viagra, for example, was developed as a cardiovascular drug, trying to reduce blood pressure in the heart. Obviously it had another function; it’s the same protein. That protein may be a target for schizophrenia down the road, you just never know. The proteins that we’ve selected, it’s really tough to know which diseases they’re going to treat.”

Proteins chosen as drug candidates have typically been good drug targets in the past, he says. He gives the example of GPCRs (G-protein-coupled receptors), of which there are hundreds with unknown functions, and which are capturing strong interest from drug companies.

“The assumption is that, well, if those ones are good drug targets, how about all these ones that we don’t know what they do yet,” he says. “They would say that would be a richer vein to tap than just your average run-of-the-mill protein, from a pharmaceutical perspective.”

Towards Drug Development

Making that transition from structural proteomics to drug development is one that Toronto, Ont.-based Affinium Pharmaceuticals Inc. is poising itself to do through its $10.5-million Viral Proteomics project.

With a goal of identifying and prioritizing targets for antiviral drug development, Affinium has three main foci to support its objective, explains Christian Burks, PhD, CSO. The early focus, he says, has been on obtaining all of the underlying cDNAs that have allowed the firm to express and purify its target proteins.

The other two foci are functional proteomics and structural proteomics, with the former geared to identifying human cellular proteins that interact with the viral proteins. For the latter, the firm has a system called ProteoVision™ that uses proprietary NMR and X-ray crystallography methods to determine protein structure.

“If you can imagine, it means starting with the individual viral protein, exposing it to the human cell and the proteins in the human cell and asking which of those proteins interact with the viral protein,” Burks says.

This could have appealing pharmaceutical value, he says, because if the human cellular proteins are known and can be interfered with in some manner, the ability of the virus to replicate and spread within the body may be disrupted. “That’s interesting from the point of view that a lot of the strategies directly interfere with the virus by developing an antibody against the viral protein,” he says.

It may also be shown that certain human proteins play crucial roles in the overall cell cycle dynamics, Burks adds, which can have relevance to interfering with particular pathways or cell functions — options that can be great targets for drugs.

“If it’s going to go in and invade a cell and perpetuate itself under the auspices of what cellular machinery exists, it over time has selected for where it can have a maximum effect of subverting or taking over the cell life cycle for the small number of proteins that it (the virus) has,” Burks explains. “But that means, to the extent that we understand which proteins the virus is interacting with, we also have insight into what are perhaps the most crucial proteins in various aspects of the cell cycle.”

Affinium is currently working on 600 proteins among a set of seven viruses: human papilloma virus, hepatitis C virus, HIV, herpes simplex virus, Epstein-Barr virus, vaccinia and cytomegalovirus. Burks says a goal is to purify the majority, and determine as many structures and measure as many protein-protein interactions as possible.

While genomics sequencing has provided a tremendously useful research resource, Burks says it has not allowed for faster or cheaper drug discovery.

“In and of itself, it (genomics) doesn’t tell you very much about whether or not a particular protein is a good target for drug discovery,” he says. “And so, proteomics steps in in a big way to really focus on proteins, what is their function, what do they do in a cell and what’s their structure, how would you be able to design or optimize a compound that would inhibit what the protein does in the cell and through all of that, having direct information about the protein is much more enabling.”

With these projects and others in the works, Canada’s research front is replete with probing protein questions — the answers to which will help solve the proteome mystery and very possibly, guide the next stage of scientific innovation.