Science & Research

Unlocking life's secrets

Manitoba researchers are breaking new ground in the study of proteins - the biochemical compounds that are essential elements in all living cells. Their work may one day lead to new treatments for diseases and conditions ranging from kidney failure to influenza.

Dr. John Wilkins says research into proteins at the U of M could lead to a host of new medical treatments
Dr. John Wilkins says research into proteins at the U of M could lead to a host of new medical treatments
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About the Manitoba Centre for Proteomics and Systems Biology

Biological facts

Bio: Dr. John Wilkins

Instrument used to decipher life's secrets has local roots

BY JOEL SCHLESINGER
Winnipeg Health Region
Wave, November / December 2011

Dr. John Wilkins is the first to admit that the four large pieces of equipment sitting in his laboratory are not the most elegant looking examples of technology ever created.

Big and bulky, with wires sticking out all over the place, it's pretty clear these gadgets, known as mass spectrometers, were not designed by the late Steve Jobs.

"Look at them," he says, half joking. "My wife took one look at one and said, 'Ugh, it looks like a beer fridge.'"

But for Wilkins, Director of the Manitoba Centre for Proteomics and Systems Biology, the beauty of these instruments - not to mention their value - lies not in their appearance. Jobs' iPod and iPad may have changed the way people listen to music or read this story, but the devices sitting in Wilkins' laboratory do much more. They unlock the secrets of life.

Originally devised by physicists and used for decades in the fields of physics and chemistry to identify and understand the nature of molecules and even smaller particles, the mass spectrometer is now being used to gain previously unattainable information about proteins, the biochemical compounds that are essential to all living things.

Not surprisingly, for devices capable of such magic, they are as expensive as they are unattractive, costing $800,000 each. The fact that Wilkins has four of them means his centre is one of the best equipped facilities of its kind in Canada.

That's no small thing. Located on the seventh floor of the John Buhler Research Centre at the University of Manitoba's Bannatyne Campus, the centre, established in 2006, has helped place Winnipeg at the leading edge of protein research, also known as proteomics.

The work is important. If DNA is the blueprint of life, then proteins are the contractors that essentially build every living cell. Understand how they operate and you can gain new insights into what happens when cells become infected or diseased.

While scientists have always known proteins were important in biological systems, it's only in the last 15 to 20 years that they started to understand the important role they play in co-ordinating how living cells operate, thanks in large measure to advances in mass spectrometry.

The beauty of the new technology is that it can identify several proteins at once in a sample. "With mass spectrometry, we can see in a single analysis several thousand proteins in one run," he says. Or, more precisely, the machines can detect chains of amino acids - or peptides - that make up proteins.

Wilkins, a professor of biochemistry and medical genetics, immunology and internal medicine at the University of Manitoba's Faculty of Medicine, says the machine is so precise it can be set to specifically detect only certain amino acid chains to target specific proteins within a sample. "You can have a very high confidence that a protein is in the sample," he says. "It's not uncommon for us to say that the odds of it being anything else are one in 10 to the 100th power."

As important as these instruments are to understanding how proteins operate, they can't do the analysis on their own. They need top-flight researchers with the knowledge, imagination and insight to understand what can be learned from proteins. And Winnipeg has a growing cadre of scientists who fit the bill. As this story is written, scientists are conducting research that may unlock the secrets of cancer, kidney disease and influenza.

"One of the biggest areas of potential use has been biomarkers," Wilkins says.

"The idea would be to take a sample from someone and determine whether it could tell us if this person has a marker that is consistent with them having cancer or some sort of inflammatory disease."

Among the more high-profile research projects at the centre that has shown potential clinical benefits for patients is a renal study for cardiac surgery patients.

Dr. Julie Ho, a nephrologist and an assistant professor in the Department of Internal Medicine at the University of Manitoba, has been working at the centre since 2006, studying the effect of cardiac bypass surgery on the kidneys.

"When patients undergo cardiac surgery, they end up on a cardiopulmonary bypass pump because they have to stop the heart to operate on it," she says. "But the body doesn't have the same blood flow as it would otherwise if the heart was working, and people can get kidney failure after that."

As part of her research, she took urine samples from patients before, during and after surgery, and compared samples of patients who did not end up with renal failure with those who did.

Using mass spectrometry, her team was able to determine that patients with low levels of a certain type of protein developed kidney failure following cardiac surgery, while those who had high levels did not. "In the heart surgery population, we actually identified a new protein called hepicidin-25 and we've been able to show it's present in patients who are at low risk of getting kidney failure," she says.

This discovery, in and of itself, is potentially helpful for improved care right off the bat because patients' urine samples can be tested for the protein in a lab immediately after surgery.

Those with the protein are at low risk of kidney failure, but those patients with very low levels of the protein or none at all are at risk, and health-care providers can at least be vigilant to look for signs of kidney trouble in this group.

Still, Ho needs to confirm her findings on a wider population base. "In order to move it into clinical practice, we have to prove that it's not just present in a small centre or population because there can be broad population changes," she says.

So far, she's only evaluated samples from the cardiac care centre at St. Boniface Hospital."That's why we're now working with this multi-centre study coming out of Yale (University)."

At Yale, a cohort study has been collecting urine samples of cardiac surgery patients from centres around the world. If the protein correlation holds true across many geographical areas, Ho says that's a good indication hepicidin-25 is a good marker for kidney failure and may play a preventive role in protecting the kidneys during surgery.

But her research doesn't stop there. The next step would be to develop a diagnostic tool that can quickly detect the protein's presence in the urine. Right now, the test involves taking samples to a lab, and that is too time-consuming for monitoring protein levels during surgery. After all, the levels of protein may be fine prior to surgery, but they may begin to drop once the heart is stopped and its role is turned over to an external machine.

"If we can somehow develop a little litmus test for the urine, where we can dip a strip into the urine and it changes colour so we can say, 'OK, this is present or absent,' that would be the ultimate goal."

While it would be the goal from a diagnostic perspective, Ho says she's also hopeful the protein itself could be administered to patients who have low levels of it to prevent kidney failure. "There are many, many steps in research that need to come before we reach that point," she says.

The first steps would likely involve lab mice, restricting blood flow to their kidneys, and taking samples of their urine and blood to determine whether injecting the animals with the protein is indeed protective. And here, too, mass spectrometry is likely to come in handy, she says, because while administering the protein might prove beneficial in preventing kidney damage, it might also cause side-effects elsewhere in the body.

Mass spectrometry could help detect those potential changes on a molecular level.

But Ho has also been working on another kidney study at the centre that involves this technology. This research aims to identify proteins that may be involved in kidney rejection in transplant patients.

"The donor kidneys don't last forever, maybe 10 to 15 years on average," she says.

Some recipients, however, experience much better results. Their donor kidney lasts 20 years or more, and, to a large extent, specialists have been at a loss as to why these patients experience optimal results while others don't. "The goal is trying to see if we can improve how long the transplant kidney will function and try to find indicators that will tell us when the kidney might fail."

She says proteomics has been instrumental in uncovering proteins that may be linked to premature kidney failure in transplant patients. So far, research has identified two proteins: monocyte chemotactic protein-1 (MCP- 1) and C-X-C motif chemokine 10 (CXCL10).

"We found in testing the urine of patients six months after getting their kidney, those patients who had higher levels of MCP-1 predicted they would likely have scarring in their transplant kidney biopsy at two years," she says. Effectively, patients with high levels of this protein after the transplant are more likely to have scarring in the kidney, and scarring is associated with premature kidney failure.

The other protein - CXCL10 - is suspected to play a role in kidney transplant rejection.

Ideally, in both cases, Ho says they would like to develop a quick and easy urine test to be able to measure the protein levels. That way when patients who are living in remote areas of the province come to the clinic, they can be quickly tested for the high levels of proteins.

"We've been able to show CXCL10 is a marker for rejection, but again, it's similar to the protein marker in cardiac patients," she says. "We need to show the presence of the protein as a marker in kidney rejection in a much broader population."

The discovery is just the beginning for Ho. The relationship of these proteins to kidney failure is still unknown, but it provides a lead to find a possible preventive therapy that could prevent failure. The proteomics centre will again play a pivotal role.

One of the key questions to be answered is whether blocking protein production would prevent kidney damage. Ho says this aspect of her research is still a long way off, and, like many other investigations into the root causes of illness carried out at the centre, it will involve the proteomics centre's shRNA libraries.

To understand the importance of the libraries, it is useful to know a bit of basic biology.

All life forms are made up of cells. Each cell contains deoxyribonucleic acid (DNA), which contains all of the cell's genetic information. This is the blueprint for making all of the necessary parts of a cell. However this information must be translated for the cell to build the products that these plans contain. This is done through working drawings in messenger RNA (mRNA) that direct the assembly of the end products, namely proteins. The mRNA acts like a construction contractor, directing cells to make certain proteins, which in turn carry out the cell processes necessary for life.

"The proteins are the machines," says Wilkins. "So they are built based upon the information that's present in the mRNA. The protein, then, is responsible for building almost everything that the cell is made up of, whether it is or isn't a protein."

In the last decade, scientists have discovered they can manipulate the activities of different mRNA using short strands of RNA, more commonly known as interfering shRNA. This specialized RNA can bind to and block the RNA from directing the manufacture of protein related to that specific part of the gene. Scientists call this a "knockdown." In effect, the shRNA suppresses or mutes the mRNA's genetic expression.

"This knockdown of protein means that the cell is now missing a part. We can now see what happens if the cell lacks that protein," says Wilkins.

This is where the centre's shRNA libraries come into play. They have 70,000 of these different types of interfering shRNA, which recognize single genes.

The technique of using shRNA to block the manufacture of certain proteins is one of the central components of Dr. Kevin Coombs' study into the influenza virus at the centre.

Coombs, a professor of medical microbiology at the University of Manitoba's Faculty of Medicine, and his team have been taking a unique approach to understanding the virus.

Normally, an infectious virus will gain entry to its host cell and hijack its genetic machinery. Think of it as an imposter, pretending to be normal RNA, the cell's regular construction contractor that directs protein synthesis.

The virus takes over that role. It directs the cell to manufacture proteins for it to replicate. This carries on until enough viral particles - known as virions - are built, and then they break out of the cell, killing it and then infecting other cells. This process repeats itself causing widespread infection until the body's immune system picks up on the viral invader and attacks the virus.

Other researchers have already used shRNA to directly affect the virus - the genes of which are made entirely of RNA - and block its ability to use the host cell's proteins to replicate. This would have the effect of stopping the virus from replicating.

By using shRNA to block a part of the virus's RNA, researchers have prevented the influenza virus from spreading throughout the body by blocking its ability to use a protein vital to replication. This has led to the development of new antivirals.

But Coombs says they have limited effectiveness. "The reason for that is because even though people are inhibiting the virus by using the interfering RNA against the virus, the viruses are still mutating and overcoming that," he says. "Even though it's a good approach, the virus is working its way around it, which is why we have to try something different."

Instead, Coombs is looking into blocking the proteins within the cell required for virus production from being manufactured in the first place, so even if the virus is able to use the protein, it's not available within the host cell and, as a result, the virus cannot replicate.

The initial stages of this study involved using a mass spectrometer to identify which proteins within the cell are necessary for the influenza virus to replicate. But they also needed to use interfering RNA to mute parts of the host cell's genes to block the manufacture of certain proteins. So far, they've identified about a dozen genes that instruct the cell to build proteins essential to the virus's replication lifecycle.

The next step is determining what happens if they "knock out" one of those genes in the cell. "Can we inhibit something in the cell which doesn't harm the cell but will inhibit the virus?" he asks. "From a therapeutic point of view, we're hoping to find there are no sideeffects to the cell and it's bad for the virus."

But the effects of muting a gene to block the production of a protein may not be without side-effects, he says.

"The problem is that the proteins required by one cell type may also be required by other cell types in the body. So if you stop cells from making a particular protein everywhere, there may be very severe consequences. In the lab, we use a few cell types to decide if something may be useful, but it may turn out that what works well in the lab could interfere with vital functions and not be a useful treatment approach," Wilkins says.

In fact, a large part of the centre's research focus is uncovering the interconnections between the genetic material and proteins in one cell with the billions of others in the rest of the body. And it's this branch of research that is the most challenging.

Finding a gene to mute in order to prevent a protein's production in a cell so you can stop a virus from replicating is complex enough, but determining which other functions within the body might be affected is exponentially more complicated.

The study of these relationships is called systems biology, and it involves complex mathematical statistical analysis to understand the multitude of potential outcomes.

Oddly enough, this technique started in an entirely different area of study, social sciences. During the Cold War, some communist states were focused on finding political dissenters through studying social interaction relationships. They created complex interaction maps that attempted to understand the vast relationships between citizens. The maps involved millions of connections, uncovering potential relationships between strangers who had never even met. That groundwork eventually led to the development of social networking that is so popular today with social media, Wilkins says.

The same concept can be applied at the cellular level, only with the added complexity that these relationships cannot be seen directly. "A protein is sort of like a snap-on tool, and each protein is thought to be able to interact with about six other partners," says Wilkins. "Those partners may determine what the protein does or where it does it, so you may redirect it."

Considering there are thousands of different proteins, the relationships can be complex and developing models will take time. But once scientists develop these models, they can help researchers more easily determine how muting the effects of one protein, or increasing the amount of another protein, will have effects beyond the intended treatment.

In the meantime, however, Coombs and other researchers continue their work in the lab at the centre. Looking through their microscopes, they pore over samples of cell cultures that have been treated with interfering RNA. They look not only for the effect on the influenza virus within the cell. They also look to make sure the cell isn't damaged.

"When we look at them under the microscope, they look perfectly fine, but we can also test them with a specific dye like a colour dye that will tell the difference between happy cells and unhappy cells," he says.

The eventual goal - finding a new class of antivirals - is still years away, he says. "It's not quite like CSI where we can solve this in an hour," says Coombs.

Like much of the other work carried out at the Manitoba Centre for Proteomics and Systems Biology, the results are leading to new insights about illness and disease. But the road to fully understanding the pathways involved in disease and some day finding new treatments is sure to be long, winding and not without its bumps.

"We've made some progress but we clearly have a ways to go still," Coombs says.

Joel Schlesinger is a Winnipeg writer.

Wave: November / December 2011

About Wave

Wave is published six times a year by the Winnipeg Health Region in cooperation with the Winnipeg Free Press. It is available at newsstands, hospitals and clinics throughout Winnipeg, as well as McNally Robinson Books.

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