A team led by Johns Hopkins Children's Center scientists has identified and successfully tamed an overactive protein that plays a key role in cystic fibrosis (CF), a genetic disorder that interferes with the body's ability to transport chloride in and out of cells.
Using a tool called RNA interference on cells in the laboratory, researchers successfully intercepted signals sent out by the rampant protein and prevented cell damage by the protein, effectively restoring the cell to normal.
"The hope is that these findings will be used to design therapies and drugs that go beyond symptom management and actually restore normal cell function to prevent CF," says senior investigator Pamela Zeitlin, M.D., a pulmonologist at the Children's Center, although she warned that they are years from developing or testing such treatments in whole animals or people. A report on the work from scientists at the Children's Center and the University of Maryland appears in the June 23 issue of the Journal of Biological Chemistry.
The overactive protein, called VCP/pr 97 (valosin containing protein), kills a chloride transporter in the cells of the vast majority of CF patients, but quieting the protein restores the cells' ability to transport chloride in and out, researchers found. The inability to transport chloride is the hallmark of CF that causes dangerous buildup of thick, sticky mucous in several organs, including the pancreas and the lungs, leading to malnutrition, chronic lung infections and lung damage.
Cells have a built-in quality-control machinery called ERAD (endoplasmic reticulum-associated degradation), which chemically "marks" defective proteins for destruction and sends them to the cell's waste-disposal complex, called the proteasome. In people with CF, defects in genes for a protein called CFTR (cystic fibrosis transmembrane regulator) interrupt the transport chemistry. Until now, researchers had not identified the precise search-and-destroy proteins that ERAD deploys to seek out the mutant CFTR.
"We were able to confirm that to get rid of the defective CFTR protein, cells deploy VCP/p97 protein, which latches onto the damaged CFTR and sends it to the proteasome for destruction," Zeitlin says. "Using RNA interference, which basically works by silencing the expression of genes or proteins, we homed in on VCP and blocked its production. That let the defective CFTR to successfully sneak past the quality control and race up to the surface."
To determine VCP's role in the destruction of CFTR, researchers compared bronchial cells from CF and non-CF patients. In non-CF cells, the protein's levels were in check, whereas they were strikingly high in cell samples obtained from CF patients.
Suspecting that inhibiting VCP would spare the chloride-transporting channels from premature demise, the team showed that when the VCP's level was lowered, it no longer destroyed CFTR.
In a second set of tests, researchers blocked the destruction of CFTR with a proteasome-inhibiting drug currently used to treat multiple myeloma. Silencing the protein by the use of RNA interference was superior to the proteasome inhibitor, researchers found.
Both the drug and RNA interference also staved off inflammation caused by cytokine IL8, which is the main inflammatory chemical produced by CF damaged cells.
"Targeting VCP, we were able to achieve two things at once -- restoring chloride channel function and curbing inflammation" says co-author Neeraj Vij, Ph.D., a postdoctoral fellow at the Children's Center. "Inhibiting specific sites in VCP can lead to the development of CF drugs."
"The goal is to develop small molecules that disrupt the binding between the VC protein and CFTR, much like tiny guided missiles that take out portions of this rampant VC protein before it latches onto CFTR," Zeitlin says.
Authors on the paper are Zeitlin and Vij, of Hopkins, and Shengyun Fang, M.D., Ph.D., of the University of Maryland Biotechnology Institute.
Founded in 1912 as the children's hospital of the Johns Hopkins Medical Institutions, the Johns Hopkins Children's Center offers one of the most comprehensive pediatric medical programs in the country, from performing emergency trauma surgery, to finding causes and treatments for childhood cancers, to delivering a child's good bill of health. The Johns Hopkins Children Center's Pediatric Trauma Service is Maryland's only state-designated trauma center for children. With recognized Centers of Excellence in 20 pediatric subspecialties including cardiology, transplant, psychiatric illnesses and genetic disorders, Children's Center physicians, nurses and staff provide compassionate care to more than 90,000 children each year. For more information, please visit: www.hopkinschildrens.org
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