Research by an interdisciplinary UW team has led to the discovery of a protein modification in animal cells that may offer a new approach to anticancer therapy.
In the late 1980s, a UW team led by biochemistry professor John Glomset and chemistry professor Michael Gelb found that certain proteins in all animal cells contain either a particular 15-carbon or a 20-carbon group of atoms, called farnesyl and geranylgeranyl groups, respectively. These "prenyl" groups are made from the chemical species isoprene, a fundamental and ubiquitous chemical building block in plants and animals. Prenyl groups seem to play a role in anchoring proteins to cell membranes, among other functions.
After Glomset, Gelb, and colleagues had demonstrated the structure of these prenyl groups, researchers elsewhere found that a certain type of protein responsible for cellular switching and growth functions, called the "ras" protein, has a farnesyl group attached to it which is critical to the function of the protein.
This finding was of tremendous interest to the scientific community because of the fact that mutated ras proteins are estimated to be responsible for up to 30% of certain human tumors. Scientists suspect that the mutated ras protein gets stuck in the "on" position, causing wild, uncontrolled growth leading to a tumor. Since the farnesyl group is essential for ras to function, if the attachment of farnesyl to a mutated ras protein could be blocked, scientists would have a way to disable the switch that turns on tumor growth. "Many pharmaceutical companies world-wide have a program to discover inhibitors of the enzyme that puts the farnesyl group onto ras," says Gelb.
This hot new field of anticancer research has its origins in several different and coverging lines of work by researchers around the world. Farnesyl groups were first discovered in the early 1980s, when workers in Japan determined that some peptides--short protein fragments--in fungi possessed a farnesyl group.
Meanwhile, ongoing research at the UW was about to lead UW researchers to the identification of farnesyl groups on proteins. Glomset had been studying cholesterol production in animal cells; he was interested in finding out when cholesterol is made during the period in which the cell starts to replicate. Glomset and colleagues found that if the cells were treated with a cholesterol inhibitor, not only did cholesterol biosynthesis stop, but the cells didn't divide any more. When the researchers then gave the cells cholesterol, the cells were not revived; but when they gave the cells mevalonic acid--the building block to make cholesterol--cell division was restored. So something other than cholesterol was responsible for arresting growth. Where did the mevalonic acid go? The researchers fed the cells radioisotope-labeled mevalonic acid, and by tracing the radioactivity, found that the mevalonic acid was incorporated into proteins. In the late 1980s, Gelb's lab became involved in the project to determine the identity and chemical structure of these attachments to the proteins.
By 1990, the UW team had identified the farnesyl group in one protein called lamin B, which is part of the membrane that surrounds the cell nucleus and gives structure to the nuclear membrane. With that discovery, Glomset, Gelb, and coworkers became the first researchers to show that farnesyl groups exist in animal cells.
Aided by the work at the UW, three research groups elsewhere working independently suggested that ras proteins also were farnesylated. Ras proteins belong to a family of small, GTP-binding proteins, that is, they bind the nucleotide triphosphate called GTP. When they bind GTP, they're activated; and when they bind the diphosphate form, GDP, they're inactivated. Ras thus acts like a switch: when a cell receives a signal to grow, such as from a growth hormone, the receptor for the growth factor activates ras, which binds GTP. Ras then activates other proteins inside the cell.
There are certain mutations in ras that prevent it from being switched off. "It seems generally accepted that a large number of human tumors can be traced to mutated ras proteins," says Gelb. There are estimates that perhaps as many as 30% of colon cancers, for example, are caused by mutations in the ras protein. "Ras is one of the more important areas for cancer research because it seems to be involved in so many human tumors," he notes.
Researchers who discovered that ras was farnesylated also showed that if they made a mutation at the site where the farnesyl was attached, so that the protein couldn't be farnesylated, ras didn't work any more. "You can take cells in culture and introduce mutated ras, causing tumorous cells to grow; then if you add inhibitor that blocks the enzyme that attaches the farnesyl group, you can watch the cells revert to normal," recounts Gelb. "In animal studies, the drug companies are feeding these inhibitors to animals in which tumors have been implanted, and they're finding some shrinkage of the tumors. Time will tell whether these inhibitors will make it into human clinical testing." Inhibitors are proprietary to drug companies, but some are peptides--short protein fragments--and some are non-peptide, natural products. Fortunately, the inhibitors don't seem to be toxic to the cells in the short term. "This is probably one of the most aggressive lines of anti-cancer research among drug companies in a long time," Gelb affirms.
About six months after determining the structure of the farnesyl group, Gelb, Glomset, and colleagues showed that a 20 carbon-group, geranylgeranyl, occurs on other cellular proteins. Among the latter are the heterotrimeric G proteins, which also bind GTP and are important in cellular switching processes, for example, in vision and in the cellular response to the "fight-or-flight" hormone, adrenaline. The UW researchers not only were the first to discover the geranylgeranyl group, they also were the first to purify the enzyme geranylgeranyl transferase, which attaches that prenyl group to proteins.
Many proteins also contain fatty acids such as the palmitoyl group, but no one has ever purified an enzyme that carries out that protein modification. As of late 1995, Gelb's group had nearly purified the enzyme that puts the palmitoyl group onto the ras protein. Given that palmitoyl is also critical to the function of ras, this finding might provide another new target for anticancer drugs, notes Gelb.