Over 90,000 people worldwide suffer from cystic fibrosis. When infants inherit two mutant CFTR genes, one from each parent, they get the condition, which results in faulty CFTR proteins on the surface of cells in the lungs and other organs.
The condition is caused by about 2,000 identified mutations in the CFTR gene, and pharmacological treatments are often tailored to each patient’s genetic profile. In the recent decade, some of these treatments have had significant success in restoring the function of the CFTR protein. However, even among patients with the same mutation, treatment response varies greatly.
Now, researchers at the University of Toronto have identified hundreds of novel proteins that may be involved in cystic fibrosis and may provide insight into why some patients respond better to current medicines than others.
Many of these proteins, which are part of a class of druggable molecules known as membrane proteins, interact with the CFTR protein, which, when missing or defective, causes mucus to build up in the lungs and other organs, which can be fatal in cystic fibrosis.
“We identified more than 400 proteins associated with either healthy or mutant CFTR, and have shown that some of them could predict the variability seen in patient symptoms and treatment responses,” says Professor Igor Stagljar, principal investigator on the paper.
“With a more comprehensive view of the CFTR protein interaction network, we can identify novel drug targets that should enable more patient-specific therapies.”
Using a platform established in 2014, researchers created a novel technique to aid in the identification of CFTR-protein interactions. The method is a high-throughput version of their Mammalian Membrane Two-Hybrid system, which enables for the identification of many more membrane proteins that interact with a given protein.
“The earlier design was array-based, and we could only screen about 200 proteins at a time,” says Stagljar, adding “With this new technology, we’ve introduced several changes that allow us to screen thousands of protein targets simultaneously, in a pooled manner.”
Stagljar and his colleagues used the technology to discover a number of previously unknown proteins, including a number of membrane proteins that could be involved in CFTR function and cystic fibrosis. Membrane proteins make up around a third of all proteins in cells and nearly half of all pharmacological therapy targets.
The Fibrinogen-like 2 protein, which is expected to play a role in hepatitis, liver illness, and immunological function, was one of the team’s most promising candidates. The researchers discovered that downregulating this enzyme causes higher expression of CFTR in organoids, which are 3D in vitro models that depict how cells interact in an organ, in this case patient-derived gut tissues.
“We think Fibrinogen-like 2 protein is a valuable drug target for cystic fibrosis, and we’re now working with our collaborators to validate other proteins that turned up in this study and in genome-wide association studies,” Stagljar adds.
While researchers have long assumed that secondary genetic modifiers and environmental factors play a role in treatment response, the new data strongly demonstrates that proteins that physically bind with CFTR are one of those factors.
“This study represents a breakthrough in proteomics and cystic fibrosis, but it would have been impossible without our many collaborators,” said Stagljar. “We developed the technology, but we’re not experts in cystic fibrosis, physiology and other fields, so we teamed up with the best and they made it happen — that’s how science works nowadays.”
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