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U Calgary s Justin MacDonald on Studying Signaling Proteins Involved in Smooth Muscle Contractility

Justin MacDonald
Assistant professor
Smooth Muscle Research Group, Department of Biochemistry & Molecular Biology, University of Calgary

At A Glance

Name: Justin MacDonald

Position: Assistant professor, Smooth Muscle Research Group, Department of Biochemistry & Molecular Biology, University of Calgary, since 2003.

Background: Research associate, Timothy Haystead's lab, Department of Pharmacology & Cancer Biology, Duke University, 2000-2002; research associate, Haystead's lab, Department of Pharmacology, University of Virginia, 1998-2000.

PhD in biology, Carleton University, Ottawa, Canada, 1998.

Justin MacDonald is planning on giving a talk next month at the Canadian Proteomics Initiative conference in Edmonton, Alberta, about using functional proteomics to study signaling proteins involved in smooth muscle contractility. ProteoMonitor spoke with MacDonald to find out more about his techniques, and about the functional importance of smooth muscle.

How did you get involved in studying signaling proteins and smooth muscle?

I did my PhD in a comparative biochemistry lab, so we were looking at how proteins are adapted in nature's strange critters — the critters that can survive in Antarctica, and undergo hibernation during winter months, and undergo extracellular freeze tolerance or anoxia tolerance. So my PhD was on signaling proteins in those animals, and how they adapted to suit their needs. For example, how do proteins work in cold? Do they still function when an animal is at two degrees? A hibernating mammal's internal body temperature will go from 37 degrees to about 5 degrees Celsius in about an hour. How does coordination signaling occur in that situation?

Then I went into another signaling lab at the University of Virginia with Dr. Tim Haystead. At that time, he was looking at mapping protein phosphatase substrates globally, but also in smooth muscle. That was around 2000.

At that time, we used proteomics to try to solve some problems that were known in smooth muscle regulation — how is smooth muscle contraction regulated? What are the kinases and phosphatases involved?

What kind of tools did you use to study protein signaling?

In Virginia, we initially were using Edmund sequencing, and then that lab was involved in methodology — coming up with ways to use Edmund sequencing to identify proteins in a high-throughput method. But the sensitivity and robustness of that technique just didn't lend itself to the sensitive nature of proteins, so the lab moved into mass spectrometry around 2002.

Using mass spectrometry, and coupling traditional proteomic technologies — 2D gels, mass spectrometry, Edmund sequencing — with more advanced things — ICAT, 2DLC, Mudpit methods developed by Yates' group at Scripps — we tried to adapt those to look at specific aspects of muscle contractility. That's what my lab does now. We're interested primarily in signaling proteins and how muscle contractility is regulated independent of calcium levels, as well as how muscle relaxation can occur — what are the kinases and phosphatases that are known to mediate these events?

What's the bigger picture that leads you to want to study the molecular mechanisms of muscle contractility?

Smooth muscle is the dominant cell type that is present in all of your hollow organs. So smooth muscle controls basic physiology such as blood pressure, birth, breathing to some degree, the GI system, the bladder. So any sort of disorder or disease that affects smooth muscle can lead to very important changes in your health and well being.

My lab is interested in muscle contractility with respect to a number of different diseases. Certainly, we're interested in the cardiovascular system and how veins and arteries function, or how they become dysfunctional.

We have a strong program through the Crohn's and Colitis Foundation of Canada looking at smooth muscle effects or dysfunction in diseases like Crohn's or colitis. Those diseases of the GI system tend to be mediated by inflammation. If there's an inflammatory insult, that inflammation is transmitted to the muscle layer and affects the motility and the ability of the gut to contract in its rhythmic and normal nature.

So we're interested in how a muscle goes from being normal and happy to being dysfunctional and unhappy.

How do you use mass spec to go about studying that?

We have a number of projects that use proteomics to some degree. The novel nature of our lab is trying to integrate proteomics with physiology. For example, we can remove very small pieces of muscle that are contractile and functional, almost in the way they would be working in your body. And we can monitor the contractility of that muscle. These are very small pieces, about the size of an eyelash. We can label that muscle with radioisotopes, or we can do antibody immunoprecipiation and try to look at complexes of proteins and signaling proteins. And then we go to mass spec to identify what they are, and whether or not the components change.

One of our projects involves looking at phosphorylation events in smooth muscle that is relaxed. Probably the most well known therapy for inducing muscle relaxation is Viagra. What that does is it induces nitric oxide levels to be increased.

There's a signaling pathway through a compound called cyclic GMP. Essentially what Viagra does it allows cyclic GMP levels to be maintained. What cyclic GMP then does is it activates a signaling protein called cyclic GMP-dependent protein kinase. That kinase phosphorylates a number of proteins in a muscle bed, which then causes relaxation.

So there's not a lot known about what the actual targets of this cyclic GMP protein kinase are, so we've been interested in mapping them.

One interesting thing about Viagra is that its target is actually ubiquitous among all muscles. However, the physiological effects of Viagra tend to dominate in the corpus cavernosum. There are some effects on the cardiovascular system, and there's a lot of work going on now to see whether or not Viagra can be used as a cardiovascular pharmalogical. But the most interesting thing there for us is why would a compound, or drug, that targets an enzyme that's ubiquitously present in all smooth muscle — this is present in bladder, in the gut, cardiovascular system, the penis — what allows specificity of signaling? We're interested in downstream events — what proteins are phosphorylated as a result of this compound? And how are they different among different smooth muscle beds?

How do you go about studying phosphorylation?

A lot of that happens prior to mass spec. The inherent problem with proteomics is the actual sensitivity and amount of protein you're going to analyze. So traditionally, what we've done is identify the protein, say a dot on a 2D gel that's phosphorylated. We identify what that dot is, and we would then try to identify what the actual phosphorylation site is. There are certainly a plethora of biochemical methods to do that. Some of them involve mass spectrometry, some of them don't. Some would involve Edmund sequencing or P32 labeling.

The methods that you would use to analyze phosphorylation will depend to some degree on the protein, and where you putatively think the phosphorylation site is. Not all proteins are the same, and therefore the techniques that you use to analyze them aren't the same.

We would use, again, a battery of techniques to try to get at where the phosphorylation site is, then downstream of that, we would probably make antibodies and then go back into different muscles and see if we stimulated those muscles, if we induce relaxation or contraction, do we see increases in phosphorylation in vivo in the muscle?

So we go from physiology — the observation of, 'You give a drug, and the muscle relaxes' — to what are the targets, or proteins involved there, to what are the phosphorylation events involved? Once we know those, we would go back and say, 'Can we show that back in the muscle, if we give this drug, that that actually happens?' In that way, we try to corroborate our results that we see in vitro back in the muscle bed itself.

What's your ultimate goal from studying these molecular mechanisms?

Well, our ultimate goal is to understand the inherent function of smooth muscle. Certainly, because of funding, you have to tie in disease to what you want to look at. But the actual aspects of smooth muscle contractility are very complicated. It's not well known what happens with signaling in normal parameters. So we're generating new data, and we're sort of putting all the pieces together in terms of how muscle contraction is regulated. With that information, you can then have a better idea of what's altered in a dysfunctional state. If you don't know what's happening in the normal state, it's hard to predict what's happening in a dysfunctional state.

We're certainly interested in identifying biomarkers of disease and potentially druggable targets for disease therapies. That would be long term. And certainly protein kinases and protein phosphatases are proteins that are the inherent regulators of just about all biological functions.

What are the major projects going on in your lab now?

One project is trying to get diagnostic markers for Crohn's disease and colitis. These diseases are chronic and relapsing. Generally, the only way now to diagnose for Crohn's and colitis is by colonoscopy — that's a fairly invasive procedure. So one goal is to able to have a diagnostic tool to assist in diagnosing patients that may have Crohn's.

Crohn's disease does have a genomic component — there's a Nod1 gene and a Nod3 gene — but those genes may be present in persons that never have active disease, so there's certainly an environmental component to these diseases as well. There's a very complicated interplay between the environmental and genetic component of an individual. What we want to try to identify is changes in protein complement of cells in the GI — in the smooth muscle, in the epithelial layer — that would allow us to determine if a patient is developing Crohn's.

We have other, more directed projects as well. One thing that happens in these diseases is the permeability of the GI epithelial layer increases, and then bacteria and other foreign substances are able to permeate through the epithelial layer and get to the lower layers of the GI intestinal wall. So we're interested in how do the proteins expressed in the epithelial layer change in these diseases. Are there specific proteins there that if changed would allow permeability to increase or decrease? So we're interested in mapping overall changes in cell surface proteins that are expressed on epithelial cells.

We're interested in some degree in how bacteria interact with epithelial proteins as well.

So those are the proteomic projects involved in Crohn's and colitis.

The other projects we have that are funded by the Heart & Stroke Foundation, and by the Canadian Institutes of Health Research, are more basic explorations of how muscle contractility is regulated. Here we're trying to identify substrates of specific kinases and phosphatases that are involved in regulating contractility in muscle.

Are you developing any novel techniques to do your investigations?

I suppose. We're not really a methodology development lab. We sort of have to adapt technologies that are under development by other proteomics labs for use in our systems. That may mean tweaking them or modifying them in some way so we can use them with our particular system.

We do a little bit of method and technology development, but that's not really our main purpose in the lab.

Do you use electron transfer dissociation technology at all for studying post-translational modifications?

No. We certainly use some new technologies, but our interest isn't in technology advancement. It's in using the technologies to address important aspects of smooth muscle contractility. I would say we're a functional proteomics lab that has integrated physiology and biochemistry tools.

Have you identified any biomarkers or potential therapeutic targets for the diseases you are working on?

The one we've been doing a lot of work on is a protein called zipper-interacting protein kinase, or ZIP-kinase. This is a calcium-independent protein kinase, and that's important because a normal contraction cycle in smooth muscle is regulated by calcium, and if you have kinases that are present that are active independent of calcium, then they, perhaps, could lead to dysfunction, if their activity is unregulated.

So we've been looking at this one protein called ZIP-kinase and trying to understand its role in smooth muscle contractility. We've been using proteomics to try to identify its specific substrates — what proteins it's phosphorylating in smooth muscle — and we would also analyze where on these proteins is ZIP-kinase phosphorylating them, and what is the functional, physiological relevance of those phosphorylation sites.

The interesting thing we can do with muscle is we can permeablize it, and the muscle retains its function in this permeablized state. We're just poking very small holes in the membrane, and that keeps all the proteins in, essentially, but allows us to introduce molecules, or drugs, that would act on the intracellular components. So in that way, we can add, say, P32-labled ATP into the muscle. We can stimulate to activate the ZIP-kinase, and then try to identify all the phosphorylation sites that would be P32 labeled.

What projects do you have in mind for the future?

One thing we're really interested in is coming up with methodologies to analyze these proteins that we're interested in, but from very, very small sample sizes. One of the problems with proteomics has been sensitivity and the ability to capture enough of the target protein to actually get sequence on. So we've been trying to come up with front-end enrichment methods to increase our ability to identify a protein by mass spec. Certainly, with our interest in Crohn's/colitis, we can get biopsy samples that are fairly small. So if we're interested in ZIP-kinase, how can we can we enrich the substrates out of that very small sample size? Certainly, there are biochemical techniques to do that. I guess one of our goals would be to improve on them and to increase specificity.

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