EVENTS | VIEW CALENDAR
Cracking the code of CFTR
LA JOLLA, Calif.—Scientists at The Scripps Research Institute (TSRI) have found evidence that a mutant protein (∆F508 CFTR) causing most cases of cystic fibrosis acquires disease-specific protein interactions that are responsible for its lack of normal function. Remodeling of these interactions explained rescue of ∆F508 CFTR function at lower temperature, and allowed the scientists to restore normal function of the mutant ∆F508 CFTR.
“The proteins and the interactions we’ve identified really fuel the pipeline for new drug targets to treat cystic fibrosis,” said Casimir Bamberger, a research associate in the lab of TSRI Prof. John R. Yates and co-author of the new study with Sandra Pankow, a TSRI staff scientist.
Pankow, Bamberger and their colleagues believe a better understanding of a protein called the cystic fibrosis transmembrane conductance regulator (CFTR) could be the key to developing new treatments. Most patients with cystic fibrosis have a mutation called ΔF508 in the gene that encodes CFTR, which keeps CFTR from folding properly and being processed correctly in cells.
In this study, researchers analyzed cell samples with a tool called Co-Purifying Protein Identification Technology (CoPIT), a new method they developed to identify proteins and analyze data. With CoPIT, they identified almost every protein CFTR interacted with, even tracking most likely secondary and tertiary protein interactions.
The results were surprising. While it was previously thought that most mutant proteins just lack one or two crucial interactions, the ∆F508 CFTR mutant had acquired an entirely new disease-specific interaction network. “Three hundred proteins changed their level of interaction, and an additional 200 proteins interacted with the mutated CFTR,” said Pankow.
“The process of making membrane proteins is complex and includes many different steps in which CFTR interacts with additional proteins,” Bamberger explains. “Thus there is a set of interactions that normally occurs for non-mutated CFTR. In case of ΔF508 deletion, misfolded CFTR protein is recognized by a different set of interacting proteins that reroute it to premature degradation, for example. In the paper, we show that recognition and rerouting can occur at many different points of the CFTR lifecycle.”
The ΔF508 mutation is a deletion mutation. The researchers’ current understanding is that the loss of phenylalanine 508 prevents correct folding of the transmembrane protein, and thus most of the CFTR protein is targeted for degradation before it even reaches the cell membrane. Based on the interactome, a small percentage of protein reaches the cell membrane despite the mutation, but does not function properly or is immediately removed from the membrane.
The interacting proteins are not malfunctioning, and neither is the interaction, according to Pankow. They are simply creating a different result. The proteins that interact with the mutated ∆F508 CFTR are different than the ones that interact with the non-mutated CFTR. Additionally, the strength of interaction can be different.
The researchers narrowed these mutant protein interactions to just eight key disruptive proteins, then used a gene silencing approach to remove or “knock down” those proteins and block the interaction of these proteins with ∆F508 CFTR. They found that without the additional interactions, ∆F508 CFTR partially returned to normal function. Bamberger tells DDNews that protein levels of interactors were knocked down using standard shRNA techniques in cell culture. If available, researchers used pre-validated shRNAs.
“The CFTR-specific anion channel function across epithelial cell membranes is improved in a standard cell culture setting, when protein levels of these interactors are reduced. Function of ∆F508 CFTR improved by 4-8 fold, or in some cases, 12-fold over control (non-treated or control shRNA treated ∆F508 CFTR cells), which is similar to the improvement seen by temperature shift to 28˚ Celsius, the gold standard in the field. The experiment does not fully recreate the situation in a patient’s bronchi and lung, though, and of course it is difficult to draw final conclusions about how well this would improve lung function in a patient,” said Pankow and Bamberger.
Interestingly, previous studies have shown that mutant CFTR regains normal function at low temperatures. Temperature shift improves the number of correctly folded and processed CFTR molecules that can eventually reach the cell membrane and improve chloride channel function. This happens at 24-30˚ Celsius. “Freezing people is not a practical treatment, of course, but this showed us mutant CFTR can be functional,” says Pankow. “So the idea behind our new study was to find new drug candidates that could mimic what we see at low temperatures.”
Researchers said the next step in this research is to look for small-molecule drug candidates that could target these disruptive proteins. The researchers have also released their raw CoPIT data publicly so other scientists can explore the clinical implications of CFTR interactions.
TSRI has been busy in other fields as well, with researchers making advances in the search for an AIDS vaccine. A new study in Immunity described four prototype antibodies that target a specific weak spot on the virus, called the V2 apex. The V2 apex could be recognized by these antibodies on about 90 percent of known HIV strains. The findings build on the success of several recent TSRI studies showing that, with prompting, the immune system can develop antibodies to neutralize many strains of HIV.
Guided by these antibodies, the researchers mimicked the molecular structure of a protein on HIV when designing their own potential HIV vaccine candidate. Two of the four antibodies did not need to mutate to bind with the V2 apex; instead, these antibodies used part of their basic germline structure, encoded by non-mutated genes. Researchers succeeded in mimicking a structure on HIV called the native HIV coat protein that let them design proteins that bind well to the germline antibodies and will hopefully start a useful immune response.
TSRI has also been researching proprioception, which tells us where our body parts are relative to each other and our environment. Now, in a study published in Nature Neuroscience, a team has identified this sensor protein in mice—Piezo2, which was found recently to mediate the sense of touch as well.
The team managed to isolate mouse sensory neurons involved in proprioception and confirmed that Piezo2 is expressed in mouse proprioceptive neurons and their muscle-embedded nerve ends. They developed two lines of transgenic mice in which Piezo2 production could be switched off in proprioceptive neurons shortly after the mice were born, and the resulting animals showed severe abnormalities in walking and limb positioning. The team found that leg muscle tissue from mice lacking Piezo2 produced almost no nerve signals in response to muscle stretching, whereas muscle tissue from normal mice produced robust signals.