|Imaging shows the normal CFTR fragment (top) curling into a helix, while the mutant fragment unravels into random shapes, blocking it from migrating to the cell surface, where it would prevent symptoms.
Folding Away Cystic Fibrosis
t’s a folding problem that any homemaker would understand. That’s what School of Medicine biochemists have discovered goes awry inside the cells of cystic fibrosis patients at the most basic level. The advance promises to speed development of better drugs for the inherited lung-damaging disease that causes about 30,000 mostly white Americans to experience ongoing breathlessness and coughing and repeated bouts of bronchitis and pneumonia.
In a report in the June issue of Biochemistry, the scientists describe the key defect in cystic fibrosis transmembrane conductance regulator (CFTR), a protein that regulates cellular salt levels and wards off bacteria.. Anyone who’s ever folded a big bed sheet, they explain, knows that one little slip can turn the whole thing into a misshapen mess. A similar thing happens in CF patients, deep within their cells. A genetic slip deletes a tiny but essential slice of CFTR.
This deletion—of a single amino acid along a chain of nearly 1,500 of them—occurs at a critical juncture in the twisting, turning protein. So instead of folding into an orderly, spiral shape, the molecule unwinds, unravels or otherwise comes undone.
Molecular traffic cops catch these misshapen molecules and keep them from passing onto the cell’s surface, where they were to shuttle chloride ions and other essentials into and out of the cell. Instead, the broken protein gets flagged, degraded and recycled.
“The deleted amino acid is like a passport,” says Young-Hee Ko, Ph.D., who initiated the project, supported by grants from the National Institutes of Health and the American Lung Association. “Without it, the protein can’t travel to the cell membrane, where it is critical for killing bacteria, especially in the lungs.” The result is that CF patients suffer a lifetime of chronic lung infections and an early death.
The key step in the work was isolating a small, manageable section of the huge CFTR molecule. Because it would have taken years to decode the structure of the whole protein in the lab, the researchers saved time by synthesizing a small section, just 26 amino acids long. They also made a 25 amino acid version that was missing the same amino acid CF patients lack. That way, they could bypass the unwieldy 1,500 amino acid structure and focus on the smaller fragments. It was like turning a 1,500-piece puzzle into a 25-piece puzzle.
Next, colleagues Michael Massiah, Ph.D., and Albert Mildvan, Ph.D., used a technique called nuclear magnetic resonance spectroscopy (NMR) to determine, or “see,” the structure of the two protein fragments. The NMR studies (see graphic) clearly showed the mutant, 25 amino acid segment in poorly structured, random shapes, and the normal segment in what senior researcher Peter Pedersen, Ph.D., calls a “beautiful helix.”
That’s when the researchers knew CF was a “folding disease”—much like sickle cell anemia—and struck on the idea of fixing the problem by correctly folding the mutant protein.
In work yet to be published, Ko used an unlikely molecule—heavy water—to fix the mutant CFTR fragment. The heavy water acts like an extra set of hands on the bedsheet and folds the defective fragment into the same helical shape as its normal counterparts. And while heavy water is unlikely to help patients because of its toxicity, researchers now have a simple “test-tube test” to screen possible new CF drugs.
“We’ve finally confirmed what we proposed eight years ago, that most cases of cystic fibrosis are a result of a protein-folding problem,” says Pedersen. “And now that we know what is broken at the most basic chemical level, we can lab test non-toxic drugs—there are millions of them that have already been studied—to see if they fold the protein. It’s the first time this kind of approach has been available.”
Francis Collins, director of the National Human Genome Research Institute, and his colleagues first identified the CFTR mutation in 1989 and subsequently cloned the responsible gene. A flurry of research followed, much of it focusing on gene therapy, which tries to correct the genetic defect in the nucleus of cells, upstream of the chain of events that make the CFTR protein. Several gene therapy clinical trials have been conducted, but Pedersen says the early over-emphasis on this approach reduced funding to study CFTR.
“One alternative to gene therapy is getting outside the cell and showing that you can correct the problem in a test tube. And that’s exactly what we’ve done,” says Pedersen. “Then you can go back and see what happens inside the cell.”