Volume 17 Number 3 FALL 2000

hats off to alcohol

A RESEARCHER'S FANCIFUL VISION OF HATS DANGLING ON HAT RACKS WITHIN OUR CELLS HELPS EXPLAIN HOW ALCOHOL PROTECTS AGAINST HEART DISEASE.

by krista conger

Daria Mochly-Rosen, PhD, has spent a lot of time thinking about hats. That much is evident from the picture that she drew and now displays, framed, on a shelf in her office. The three-part, somewhat faded, pencil drawing shows a cowboy hat, a child's beanie and a top hat swooping across the paper, each in search of its own custom-shaped hat rack. The racks themselves sprout incongruously from the walls and ceiling of an odd-shaped room.

The picture depicts an idea about the inner workings of cells that occurred to associate professor of molecular pharmacology Mochly-Rosen over 10 years ago when she was starting out as an assistant professor at the University of California, San Francisco. In the drawing, the hats represent members of a certain protein family, and the hat racks act as homing beacons that summon each family member to specific areas within the cell. Much like rooms in a house, a cell is divided into specialized compartments. The racks function to limit the protein's activity to a particular room.

Although it seems like a fairly simple idea, it took years for Mochly-Rosen, now Stanford's Reed-Hodgson Professor in Human Biology, to convince her peers that the hat racks shown in her drawing actually exist. When she drew the picture, she fully expected her efforts to help illuminate the mechanisms at play within our cells -- in particular, the cells of the heart, which are rich in these proteins. What she couldn't have predicted, though, was that her attempt to prove the existence of these "hats and racks" would reveal new pathways with potential to help people with heart disease live longer.

A visitor's impression of Mochly-Rosen's office on the third floor of Stanford's new Center for Clinical Sciences Research building is one of light and space. Small, brightly colored glass bottles and vases, and stained glass reproductions of modern art glow against a curved wall of windows. Shelves lined with scientific texts and copies of journal articles also host pictures of her four children, smiling at the camera.

Seated at a round table, Mochly-Rosen tells about the Tuesday night in 1996, when she realized that after pursuing hats and racks for 10 years, she had identified a tiny protein fragment that might protect people from heart attacks. "I literally jumped out of my chair," she says. "I knew that I had found an activator, and that it would be very special."

The activator that Mochly-Rosen had been looking for was a small molecule that could nudge a larger protein, or hat, to hook onto its designated hat rack. Hanging on its rack, the protein might remain active longer.

The larger protein that Mochly-Rosen was studying was a common enzyme, protein kinase C, or PKC. PKC is a key player in controlling how a cell responds to external messages telling it to multiply or to morph into a new type of cell. Mochly-Rosen believed that the activator she had just discovered would help her dissect the roles played by the several forms of PKC that can occur within one cell.

Mochly-Rosen's lab members, though, were not so quickly convinced of the activator's importance. Despite Mochly-Rosen's persistant urging, they were not eager to pursue the late-night finding.

"Nearly everyone who worked on the project had to be forced into it. And although I want people to argue with me when they don't agree with my ideas, this time I knew I was right," she laughs.

Mochly-Rosen has had plenty of experience arguing and being right. As far as the general scientific community was concerned, when Mochly-Rosen drew the hat picture in 1988, the hat racks were only a figment of her imagination. Most experts in the field thought that a physical modification of the proteins themselves was responsible for determining when and where each family member carried out its functions.

Mochly-Rosen was not discouraged by the initial lack of acceptance of her theory. In fact she regarded its low profile advantageous: It protected her from much of the competition that dogs scientists who are working on a popular topic.

"It was a major benefit that our working hypothesis was not accepted," she says. "I really think I was blessed; I was given a lot of time to work on it."

That time started in 1984 when Mochly-Rosen joined the lab of biochemist Daniel E. Koshland Jr., PhD, at the University of California, Berkeley, as a postdoctoral researcher, after emigrating from Israel. Koshland inspired her to think "out of the box," she says, and to focus her interests on the big picture. In that spirit, she began her studies of how PKC helps to transmit signals from outside the cell to the internal "movers and shakers" of cell control.

Like a relay runner, inactive PKC loiters in the cell's cytoplasm, waiting to be tagged by an upstream protein. When the signal comes, PKC moves to the membranes of the cellular compartments and activates nearby protein partners, or substrates, by tacking on a phosphate molecule. The molecular brigade amplifies the initial signal by recruiting more molecules at each step of the process and allows the cell to respond quickly to changing external conditions.

Mochly-Rosen became interested in how the slightly different forms of PKC know to migrate to the correct locations inside the cell and how they manage to pick their partner protein out of the medley of potential substrates. She speculated that individualized receptors anchor the activated PKC near its target molecules and limit its range of activities -- much as place cards at a dinner table determine the identity of one's dining companions and steer the direction of conversation. She named the receptors RACKs (for receptors for activated C kinases) and drew the hat picture that sits on her office shelf today.

At the time, however, because experts in the field were able to show in a test tube that PKC can associate with the membranes without any additional help from other proteins, they were reluctant to add yet another layer of complexity to an already multi-tiered system.

"RACKs went against the dogma," she says. "Nobody paid any attention to me because it seemed that I must be wrong."

It took three years of research to isolate the first RACK from rat heart cells and three additional years to show that it bound to only one form of PKC -- a project carried out by postdoctoral fellow Dorit Ron, PhD, now an assistant professor at UC San Francisco. The next challenge was to show that binding to its respective RACK is required for the proper, specific activity of each form of PKC. Mochly-Rosen planned to do that by finding ways to inhibit or stimulate PKC's trek from the cytoplasm to the membranes while simultaneously monitoring what happened in the cell.

In order for Mochly-Rosen's plan to work, she had to pick a form of the protein she could easily observe changing from its inactive state to its active one. Former postdoctoral fellow Marie Helene Disatnik, PhD, helped show that while they could follow the movement of several of the family members, epsilon PKC molecules, found in heart muscle cells, advertise their transition the most clearly, by moving from seeming chaos into line formations throughout the cellular soup.

Now, using epsilon PKC molecules (abbreviated ePKC), Mochly-Rosen was ready to start teasing apart the individual roles played by the different PKCs in the heart cells. If she could run interference between ePKC and its receptor -- and if her RACK theory was correct -- she would be able to selectively prevent ePKC from carrying out its normal function without affecting any of its siblings in the PKC family. Conversely, if it were possible to enhance the binding of ePKC to its receptor, she should be able to follow its unique activity even in the absence of an external signal.

To carry out her plan, Mochly-Rosen would try to muck up the binding strategy used by all of the various PKC molecules. Folded back on themselves like hairpins, the inactive forms of PKC conceal their sites of interaction both for their RACK as well as their substrate molecules. But when external signals activate them, the molecules unfold, revealing their RACK binding site and starting their migrations from the cytoplasm to the intracellular membranes. Once there, they can begin activating their partner proteins.

Mochly-Rosen knew that if she could design a small peptide that mimicked the three-dimensional topography of the spot on ePKC that binds to its RACK, she might be able to keep ePKC from fulfilling its function simply by blocking all its binding sites. The principle is similar to covering the electrical outlets in your house with plastic pronged safety covers. When all the outlets are covered, there is no way to plug in your lamp, your hair dryer or your computer.

As they had hoped, when Mochly-Rosen and John A. Johnson, PhD, a former postdoctoral fellow, constructed such a molecular copycat for ePKC and introduced it into rat heart cells, they were indeed able to block the interaction between ePKC and its receptor. And, although it was more difficult to see the effect, they were able to repeat the trick with several other forms of PKC in the heart cells.

But analysis of these cells also revealed a surprise, shedding light on the scientific debate over how the heart responds to the lack of oxygen resulting from a heart attack.

PREPARING FOR OXYGEN DEPRIVATION

In 1986, researchers studying the effect of oxygen deprivation on the heart muscles of dogs found something curious. If the heart is exposed to a brief period of ischemia, or oxygen deprivation, and then allowed to recover, the extent of the damage done during a later, longer challenge can be decreased by as much as 25 percent. Individual heart cells fared even better in subsequent studies carried out in Mochly-Rosen's lab. Up to 50 percent more cells were able to survive a severe oxygen deprivation when it was preceded by a brief ischemic period. It seems that a preview of the horrific coming attractions protects against future damage. Researchers called the perplexing effect preconditioning.

Strangely, long-term, moderate alcohol intake seems to have much the same effect as preconditioning. Populations who tend to drink regularly with meals, like the French, have lower rates of cardiovascular disease. And many studies have identified links between moderate alcohol consumption and reduced rates of cardiovascular disease. Some of the scientists in Mochly-Rosen's lab are trying to understand the alcohol/heart health connection (see sidebar).

Although scientists don't yet fully understand the molecular reasons behind the protective effect of both preconditioning and alcohol consumption, they do know that in both cases PKC is required to shield heart muscle cells. But until Mochly-Rosen and her lab members began tinkering with the interaction between ePKC and its RACK, it was impossible to distinguish which of the many forms of PKC found in heart cells is the primary molecular player in this effect.

Former postdoctoral fellow Mary Gray, MD, and collaborator Joel Karliner, MD, chief of cardiology at the Veterans Affairs San Francico Health Care System, performed a series of experiments confirming that the ability of ePKC to migrate to the intracellular membrane upon activation correlated with the protective effect of preconditioning. In other words, if ePKC couldn't bind to its receptor, preconditioning was no longer able to protect the cells from longer periods of oxygen deprivation. In contrast, blocking the binding of other family members to their RACKs didn't affect the benefit of preconditioning.

Although the inhibition experiments were interesting, Mochly-Rosen was even more excited about finding a molecule that could keep ePKC active all the time. If ePKC were solely responsible for protecting heart muscle cells, keeping it in a constant state of activation could be life saving for someone with a high risk of heart attack.

The researchers thought they might create such a drug by again using their technique of molecular mimicry to fool PKC into assuming its open conformation. But this time they wanted to try to create a molecule that would masquerade as, but not match exactly, a very small piece of a RACK that binds to PKC. Although the full-length PKC has to wait for upstream signals to pop open the molecule, they thought that the tiny "pseudo-RACK" peptides might be able to wiggle inside the bend in the inactive PKC molecule and find the RACK binding site. Once there, the binding of the small peptide might disrupt the forces that keep the inactive molecule folded upon itself and allow it to flop open even without the upstream go-ahead. They hoped that when PKC was fully extended, the true, full-length RACK could displace the imposter, bringing PKC to its waiting substrates.

This is the activator that Mochly-Rosen was searching for that Tuesday night in the lab four years ago. The molecule that the lab has designed based on that finding is the most promising PKC modulator known. Experiments by research associate Tamar Liron have shown that introducing the small eRACK-like molecule (dubbed pseudo-eRACK) into isolated heart cells causes about twofold more ePKC than usual to move to the intracellular membrane and bind to its RACK. The pseudo-eRACK-containing cells were also almost twice as likely as the control cells to survive a long period without oxygen. Perhaps even more important, mouse hearts expressing the pseudo-eRACK were better able to recover from an ischemic event than normal hearts, and their release of an enzyme indicative of cellular damage was decreased by more than 60 percent when compared with controls.

"It looks really good," says Mochly-Rosen. The fact that the peptide also provides some protection even when injected into the heart after the ischemic event has already occurred -- as shown recently by graduate student Leon Chen -- may provide hope to people suffering from cardiovascular disease, whose pending heart attacks obviously can't be predicted.

In fact, the results in mice were so encouraging that Mochly-Rosen has established a collaboration with Felix Lee, MD, a fellow in cardiology at Stanford, and Paul Yock, MD, professor of cardiovascular medicine, to test the peptide in pigs sometime this summer. Together the researchers plan to use both the inhibitor and the pseudo-eRACK with signal sequences that will allow the peptides to be taken up into the heart cells when injected into the bloodstream. They plan to monitor the peptides' effects on the heart cells' responses to periods of oxygen deprivation. Mochly-Rosen and Lee are particularly interested in how the heart cells will respond to the activator, pseudo-eRACK.

"I think we're going to find that we will see cardioprotection," Lee says. "In mice and rats the pseudo-eRACK looks very protective." If the researchers see similar results in the pigs, they plan to design a clinical trial for humans. Mochly-Rosen envisions a future in which a heart attack patient entering the emergency room could receive pseudo-eRACK through a catheter directly into the heart, just as anti-clotting agents are administered today.

An even better alternative for a person with cardiovascular disease would be an oral therapy that could be taken once a day like a vitamin. Mochly-Rosen is currently working with Paul Wender, PhD, professor of chemistry at Stanford, to turn the peptide into a therapeutic compound -- no easy task since the molecule must be able to weather the harsh conditions of the digestive system and be efficiently assimilated by the cells that need it most.

Although exploring the clinical implications of pseudo-eRACK is important, Mochly-Rosen has not abandoned her quest to understand how the different forms of PKC exert their effects on heart cells. She and her lab members are trying to identify which of ePKC's substrates gain extra phosphate tags in the presence of the activator. Those molecules may be the other downstream runners that deliver the protective message.

"We are casting a large net, hoping that we will come up with something," she says.

In addition to mice that express pseudo-eRACK, Mochly-Rosen and her lab have generated mice that express the inhibitor of ePKC in their heart cells. This effort, carried out in collaboration with Gerald Dorn II, MD, chief of cardiology at the University of Cincinnati, has revealed that the hearts of mice expressing pseudo-eRACK have thicker walls but still function normally. In contrast, mice expressing the inhibitor peptide die within 30 days after birth. The hearts of these sick mice are larger than normal, with very thin walls.

Mochly-Rosen has also begun to investigate the role played by another form of PKC in the heart cells -- deltaPKC, or PKC. Preliminary studies by graduate student Chen have shown that PKC plays yin to ePKC's yang: PKC pushes damaged cells toward death, while ePKC pulls them back from the brink. Having two opposing forces helps the cell to respond quickly and appropriately to external signals.

Mochly-Rosen's RACK theory has led her down some interesting paths, she says. Grateful for the support of the National Institute of Heart Lung and Blood and the National Institute of Alcohol and Alcoholism, she plans to follow the paths to their ends. But now that her theory is more accepted she sometimes misses the old days, when she had only her hat picture to consult while planning her experiments.

"It is really a luxurious position, working like that," she says, "but I think that's over now. Maybe I need to find another project that goes against the dogma."

Pull another unexpected theory out of a hat -- and prove it? Now that would be a trick. SM

Related story:
A Toast to Heart Health