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, 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," said senior investigator Pamela Zeitlin, a
pulmonologist at the Children's Center and professor in the
School of Medicine, adding that the researchers 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) that
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 said. "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
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 in cell samples obtained from CF patients,
they were strikingly high.
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," said co-author Neeraj Vij, a
postdoctoral fellow at the Children's Center. "Inhibiting
specific sites in VCP can lead to the development of CF
drugs."
Zeitlin said that the team's 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."
Authors on the paper are Zeitlin and Vij, of Johns
Hopkins, and Shengyun Fang, of the University of Maryland
Biotechnology Institute.