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A Sense of Wonder

e calls it basic research. Gary Bokoch, Ph.D., a professor and immunologist/ cell biologist at The Scripps Research Institute (TSRI), uses the term "basic" to describe his work with Rho GTPases, a family of proteins that regulate various processes that all cells, including white blood cells, need to function.

Basic the research may be, obscure it is not. When activated by chemical signals like hormones or cytokines, GTPases prompt white blood cells to protect the human body against invading pathogens. Without GTPases, our defense against invading microorganisms wouldn't function or would be in shambles.

GTPases, in fact, are indispensable to the regulation of almost any type of complex cell function. Acting as molecular switches in a variety of physiological systems, they control such key processes as changes in the cell's cytoskeleton, cell growth, and cell death.

Still, GTPases are not a subject you're likely to stumble across at your next dinner party. Like anything in science, discussing these proteins can sound as simple as a spring day or as complex as an explanation of the precise function of neutrinos. Bokoch often thinks of this research topic both ways, usually with a touch of amazement in his voice.

"These proteins are basically switches. When they bind to [guanine nucleotide] GTP, they're active and things happen. When they bind to GDP [another guanine nucleotide], they become inactive. When they're active, they regulate very fundamental events like enzyme activity. But because there are so many of these GTPases acting on so many fundamental processes--more than 20 members of the Rho family have been identified so far--the operation and regulation of the system can quickly become very, very complex."

A lot like explaining neutrinos, come to think of it.

A NEW WORLD

Bokoch first worked with these unique and elegantly simple proteins when conducting postgraduate work with Alfred Gilman, a future Nobel Prize winner and one of the men responsible for their discovery. Gilman, along with fellow researcher Elliott Ross, was a co-discoverer of the G protein--another name for GTPases due to their binding of and regulation by nucleotides GTP and GDP--purifying the first one in 1980. For his work on the discovery of the G protein and its roles in hormonal signaling, Gilman shared the Nobel Prize in Medicine or Physiology with Martin Rodbell in 1994.

Bokoch had pursued his graduate work at Vanderbilt University in Tennessee studying leukocytes on what he calls the "functional level." This involved research into intracellular signaling, a field the university was famous for worldwide. Gilman had taken a novel approach to understanding signaling--duplicating the actions of these signal-transducing proteins in the laboratory instead of trying to observe them in vivo. It was that scientific activism that brought Bokoch to the project.

"It opened up a new world for me, discovering these proteins, and trying to understand how they worked in a laboratory setting," Bokoch says. "I wanted to do the same thing with leukocytes [another name for white blood cells], so we could finally understand how they worked at a biochemical and molecular level. Leukocytes are amazing cells in that they're almost like independent creatures. They move around, they engulf other cells--they eat them, really--and they respond rapidly to things that you do to their environment. Yet it was always clear to me that these activities were ultimately the result of highly organized, yet astonishingly adaptive, signaling processes."

On the other hand, he says, white blood cells are somewhat delicate. Their lifetime is measured in hours, so that the body must produce millions of them each day to provide the necessary level of immune protection. What they lack in lifespan, they make up in ingenuity. Smaller than most cells (one of the reasons that they're so difficult to work with in the laboratory), they are flexible enough to squeeze through the spaces between other cells. Like tiny mutant warriors, they distort themselves, slip through cell layers, and pop up on other side, ready to attack the next pathogen they meet.

About this time, Bokoch came across a series of papers from a group headed by Professor Charles Cochrane, M.D., at TSRI. It turned out that the TSRI researchers were also interested in the biochemical qualities of leukocytes, the very topic that Bokoch was hungry to study.

"They were advertising for somebody to help with their work," he recalls. "I was fascinated by what they were doing, and that was the thing that brought me here." Bokoch had intended to stay on the East Coast, but the chance to expand his studies of GTPases and leukocytes at TSRI proved to be stronger than the pull of geography.

COMPLEX AND FASCINATING

When Bokoch first went to work at TSRI, his main interest was in the big GTPases that receive signals on the cell surface. But Bokoch, along with other researchers, began to recognize that there were other, smaller types of GTPases, although little was known about their biochemistry or their function. His work led to the discovery in 1991 of the first known function for a small GTPase--regulation of the formation of toxic oxygen metabolites (oxidants) by white blood cells.

Since then, his laboratory has focused on understanding the complexities of cellular regulation by the Rho group of small GTPases, as well as continuing studies on leukocytes, especially their ability to chemotax and to generate oxidants to kill microorganisms. Curiously enough, one form of these oxidants is hydrogen peroxide, and another is related to household bleach.

"That would be a significant benefit of our research, since [a large number] of patients who see a doctor are being treated for problems related to inflammation. Also, the recent realization that oxidants function as signaling molecules in non-leukocytic cells suggests our studies may have implications for other diseases as well."

It soon became apparent to Bokoch and his colleagues that these small GTPases were involved in other cell processes, including the regulation of the cell cytoskeleton, which determines the ability of cells to move, and to change shape and function. In the case of cytoskeletal regulation by Rho GTPases, one of the primary components is a kinase with the name of PAK. Kinases are enzymes that transfer certain chemical groups between molecules.

The PAK enzyme, in conjunction with other proteins, allows the cellular cytoskeleton to modify its shape--to shape shift, in other words, like a fictional superhero. The implication of this for such critical processes as inflammation, angiogenesis, nervous system development, and cancer metastasis is another area where Bokoch's GTPase research connects with the therapeutically significant.

His laboratory's intensive studies of how Rho GTPases regulate the intricate machinery of the cell cytoskeleton, Bokoch explains, also provide insights into the innate immune response against bacterial and viral infection.

If Bokoch is changing the way medicine views the immune system, he's also part of a revolution in technology that allows him and his colleagues to understand the mechanisms of cell signaling by watching them in real time.

"It became clear very quickly [a few years ago] that an important aspect of cell signaling is where and when it takes place within the cells," he says. "[Until recently,] we had no good ways to observe that. At Scripps, several people were developing fluorescent indicators to look at signaling in real time, and we've tried to incorporate that into our work. In a paper published this year with our TSRI colleague Klaus Hahn, [Ph.D.,] we look at the actions of Rac in human leukocytes with a fluorescent probe. We can see Rac turned on at very specific places within the cell and can observe how the dynamics change constantly during a complex process such as chemotaxis."

FROM SPIDERMAN TO SCIENCE

A fan of X-Man comics where one of the more attractive mutants is also a shape shifter, Bokoch also has a ragged edition of the first Spiderman comic. Like Spiderman--a.k.a. Peter Parker--Gary Bokoch's life has involved some transformations.

Born on the hardworking shores of Lake Erie, Bokoch's father was a policeman, his grandfather a miner who spent his life in the coal mines of western Pennsylvania. Bokoch started out catching snakes and frogs in the nearby woods and began his life-long habit of reading science fiction, a habit that got him thinking about actual science.

He built science projects in his basement, Van de Graaff generators, and an electrical carbon arc furnace--a contraption that produces an electrical arc between two carbon rods that he used to melt nails and, on more than one occasion, short out the entire electrical system of his parents' house. Like others of his generation, he was fascinated by the ongoing space program and kept a file of newspaper clippings.

"As I got deeper into the idea that I might want to be involved in science, I also had a couple of high school biology teachers who made me excited about biology," he remembers. "One even had a Ph.D., unusual for a high school teacher."

From there it was a short leap to college chemistry and biology, and trying to figure out a way to combine the two, which he accomplished with a Ph.D. in pharmacology. In the end, he arrived at TSRI, an institution that would seem custom made for anyone interested in seeing how that combination of sciences might actually work.

"The thing that I like most about Scripps," he says, "is its tremendous academic environment. There's interesting work going on, and since everybody is excited about their work, we all look for ways to interact with each other. Collaboration is encouraged, and there's a real sense of camaraderie, especially within the department. Because so many biological systems depend on GTPases, we connect with a lot of people."

As a scientist, Bokoch has managed to hang onto his own sense of surprise and wonder about what he does, in and out of the lab.

"I'm constantly amazed by these incredibly intricate biological systems. How they evolved is just stunning to me. If you try to rationalize how things got that way, you can only come away with a sense of awe, not to mention a certain feeling of humility."