Last year, I turned 30. It’s one of those numbers that often causes humans to pause and think about what we want to accomplish in the next decade of our lives – run a(nother) marathon, travel the world, land an awesome job… But how many of give much consideration to the question of if we’ll be around to celebrate that next decade? For patients with cystic fibrosis, an inherited disease that affects the lungs and other organs, that if is a big one – the median survival age for cystic fibrosis patients is 38.
Cystic fibrosis is an incredible example of a truly molecular disease. It’s caused by mutation of one gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a protein that helps maintain salt homeostasis in epithelial cells. Epithelial cells line surfaces and cavities of our bodies, things like the airway, sweat glands, and ducts in the liver and pancreas; these cells stick close together to create barriers between different environments and maintain a careful balance of fluid and electrolyte shuttling between these environments. CFTR transports chloride and other anions into mucus lining the epithelium, and sodium and then water follow to maintain normal, fairly fluid mucus. If CFTR is dysfunctional, the balance breaks down; thick mucus builds up in passages of the airway and digestive system, which become blocked. The obstructions alone cause serious respiratory and gastrointestinal problems, but the thick mucus in the airway also creates a cozy home for bacteria, setting the stage for respiratory infections. The mucus and associated complications significantly impact quality of life, but it’s the infections that typically kill.
CFTR is a massive and complex protein, that must be properly folded, embedded in a membrane, and shuttled to the cell surface. The fully functional, or mature, form weighs in at almost 190,000 Daltons (g/mol for the chemists out there ), which is more than 3 times the size of a typical protein. It’s initially expressed as a smaller precursor protein (~135,000 Daltons), but as CFTR passes quality control checkpoints, it’s decorated with sugar chains that tell cellular machinery that this CFTR protein is OK and ready for the next step of the process. With normal CFTR, only about 30% of CFTR that’s expressed actually transits the checkpoints successfully. The remaining protein is trashed without ever having done the job it was made to do. Just as sugars flag proteins that have passed quality control, ubiquitin commonly flags proteins that need to be degraded. Ubiquitin is a small regulatory protein that can be attached to a specific group (lysine) on other proteins. Ubiquitin can be conjugated to itself to create poly-ubiquitin chains, which target the proteins to the proteasome, a large multi-protein cylinder that cuts proteins into smaller pieces, or to the lysosome, an acidic intracellular sack filled with degradative proteins; in both cases, the end result is destruction of ubiquitinated proteins. At Experimental Biology 2013, Dr. Seakwoo Lee, a research fellow in Pam Zeitlin’s lab at Johns Hopkins Children’s Center, presented work on how ubiquitin modifies and regulates CFTR stability.
The Zeitlin lab worked with Michelle McClure in Eric Sorscher’s lab at the University of Alabama at Birmingham to use mass spectrometry (“mass spec” to its friends) to identify residues of CFTR that had been modified by ubiquitin used. CFTR was isolated from cells and chopped into smaller fragments; this process removes ubiquitin but leaves behind a trace of it (glycine-glycine) where the ubiquitin tag once resided. Mass spec defined the amino acid sequences of fragments and the locations of glycine-glycine modifications and thereby ubiquitin. Lee mutated each glycine-glycine tagged site to arginine, a substitution that maintains the charge of the protein but that cannot be modified by ubiquitin. For each mutant, he looked at the protein expression levels of total and mature CFTR. He determined whether ubiquitination of each lysine targeted CFTR to the proteasome or the lysosome by using small molecule inhibitors. Because CFTR only functions at the cell surface, Lee checked surface expression of the mutant by confocal microscopy and Western blotting. Finally he checked the ability of mutants to modulate expression of the inflammatory chemokine interleukin-8 in an epithelial cell line.
They defined seven specific sites of modification, scattered throughout multiple domains of the protein. In all but one instance, lysine->arginine mutations increased the amount of total CFTR protein and, importantly, the amount of fully matured protein. Addition of ubiquitin to different CFTR domains targeted the protein to different pathways for destruction. Modification of the N-terminal and nucleotide-binding domains targeted CFTR to the lysosome, whereas modification of the regulatory domain targeted CFTR to the proteasome. Now you might think that more CFTR and more mature CFTR would also mean more CFTR on the cell surface where it’s needed to function. Yet mutants that prevented proteasomal degradation were actually expressed at lower levels than wild-type CFTR on the cell surface, even though these mutations produced more mature CFTR inside the cell.
So Lee saw that CFTR expression was stabilized by introducing mutations that prevented ubiquitination, but he wanted to find out if it functioned properly. Previous studies had shown that CFTR surface expression and activity suppresses basal inflammatory signaling. In cultured epithelial cells, dysfunctional CFTR activates the transcription factor NFΚB, which goes to the nucleus and turns on genes associated with inflammation. Co-expression with functional CFTR counteracts this program. Lee used one of those inflammatory program genes (interleukin-8 or IL-8) to look at the functional outcomes of his CFTR mutations when co-expressed with a completely non-functional CFTR. Mutants expressed on the cell surface at levels approaching wild-type were non-inflammatory. With mutants that failed to localize to the surface, cells still produced
elevated IL-8. However, lysosomal inhibition increased surface expression of these mutants and decreased IL-8 synthesis. How did that happen? Lysosomes are tightly linked to endocytosis – that is, internalization of things bound to or embedded in the surface membrane. Like many surface receptors, CFTR is rapidly internalized and shuttled back to the surface (recycling) or shipped to the lysosome for degradation. Lysosomal inhibition ultimately piles up and inhibits endocytosis. Lee’s result might suggest that stabilizing CFTR expression alone is insufficient for boosting CFTR activity. Instead drug discovery and development might also need to CFTR internalization and recycling.
The FDA recently approved the first drug that actually treats the molecular cause of cystic fibrosis. However, this drug addresses only one aspect of CFTR dysfunction, its direct activity. Over a thousand mutations have been identified in cystic fibrosis patients, and mutations that alter CFTR stability and localization present more of a challenge for therapeutic targeting and will likely require a combination approaches. One day, researchers might be able to leverage the sites and roles of CFTR ubiquitination to develop a drug to enhance CFTR surface expression. However, Lee’s data are preliminary, and further studies are needed to confirm the mechanisms of ubiquitin regulation of CFTR. The Zeitlin lab also needs to identify small molecule modulators of the process and define the therapeutic benefit and limits of this approach. In the past few decades, the survival age of cystic fibrosis patients has increased dramatically, but there’s still a long way to go.