CFTR and Ion Transport

Proximity Ligation Assay(Proximity Ligation Assay detecting GFP-F508del CFTR (green) in CFBE41o- airway cells, nuclei (DAPI blue). Red dots: CFTR-SUMO-1)

Biogenesis and degradation of WT and mutant CFTR alleles (Aridor, Bertrand, Brodsky, Bruchez, Frizzell, Pilewski, Thibodeau): The group studying pathways of protein folding vs. degradation uses the P30 cores to evaluate the role of CFTR’s structure and chaperone networks in WT and F508del CFTR biogenesis and degradation. More recently, the group has expanded to examine CFTR2 alleles linked to disease. Model cells and airway cell lines are used, with an emphasis on patient-derived primary airway cultures (Core A).  HBEs continue to provide the most relevant pre-clinical model for translational studies of CF. This team has participated in target protein analysis and validation in collaboration with both academic labs (Bill Balch, Scripps; Harvey Pollard, USUHS; Scott Blackman and Garry Cutting, Johns Hopkins; Eric Sorscer, Emory, Gergely Lukacs, McGill; Phil Thomas, UTSW; Fei Sun, Wayne State and with other members of the CFTR Folding Consortium), and industrial partners (Vertex, Proteostasis Therapeutics, Pfizer and the Flatley Discovery Lab). The success of this group has relied on materials and methods from the Airway Cells and Tissues and Assays Cores (A&B).

   Structural characterization of CFTR: The Thibodeau lab has been involved in the purification and structural characterization of CFTR using biochemical and biophysical approaches.  His initial studies focused on the production and structural characterization of the cytosolic domains of CFTR, including NBD1, NBD2, and the R-domain, and are now expanded to include expression and purification of full-length WT CFTR and mutants as part of the CFFT CFTR 3D Structural Consortium.  These highly-purified reagents have been used for biophysical studies with academic collaborators (Ray Frizzell, Jeff Brodsky, Jen Bomberger, Pitt; Bob Bridges, Rosalind Franklin; and members of the CFTR Structure Consortium) and have been provided to industrial partners for small molecule mechanism of action (MoA) studies.  The ability to produce these reagents enables a variety of in vitro studies, which now include cryo-EM and X-ray crystallography for the determination of additional CFTR structures, biochemical studies to evaluate ubiquitin and SUMO post-translational modifications, and solution-based spectroscopic studies to evaluate protein dynamics and energetics.  The lab has developed in vitro methodologies to examine small molecule binding for pre-clinical (MoA) studies.

   SUMO pathway augments CFTR biogenesis:  This effort grew out of a Frizzell/Brodsky lab collaboration starting with a yeast screen that identified an Hsp27 initiated pathway in CFTR degradation by its SUMO-2 modification. Subsequently, a Protoarray identified PIAS4, a SUMO E3 enzyme, which conjugates CFTR with SUMO-1, increases CFTR biogenesis, reduces F508del ubiquitylation augments corrector impact. We now find that PIAS4 markedly stabilizes more than 20 rarer CFTR2 mutants, which can be divided into groups quantified their abilities to respond to folding correctors (e.g. VX-809), and suggesting that different corrector compounds may be needed to provide therapy for specific CFTR mutations.

   Development of ubiquitination inhibitors:  Polyubiquitinated F508del CFTR accumulates when proteasome activity is blunted; therefore, preventing ubiquitination should facilitate F508del-CFTR biogenesis, especially when combined with folding correctors. These data encouraged the Brodsky group to test whether a small molecule inhibitor of the ubiquitin pathway would improve the ability of folding correctors to restore F508del CFTR biogenesis and function. Indeed, a modest inhibition of the ubiquitination machinery, in combination with a folding corrector, synergistically rescued F508del-CFTR, and in collaboration with Pfizer, ~25 analogs were screened, some less toxic and more active.

   In a related project, cell impermeant fluorogens have been used by the Frizzell group to quantify F508del rescue by a panel of CFTR correctors and their impact on the PM stability of WT and F508del CFTR (i.e. recycling vs. degradation). The Assays Core is implementing the FAP technology for the CFTR2 project (, to determine corrector rescue efficacy and cell surface stability of CFTR2 mutants using CFFT panel correctors ( Our CMU colleagues, who continue to evolve fluorogen-FAP chemistry, have established a new company, Sharp Edge Labs, which performs CFTR corrector assays for biotech companies based on our work ( In fact, they are under contract with several biotech firms for secondary CFTR drug screens.

   Bench-to-bedside: Our contribution to the development of Ivacaftor and Lumacaftor resulted from the membership of Dr. Frizzell on the CFFT-Vertex Collaborative Committee and his inclusion in the authorship of the associated publications. In addition, the P30 Human Airway Cells Core provided HBE cells to Vertex, facilitating their analysis of Ivacaftor in HBE cells with a G551D/F508del genotype.  Similarly, pre-clinical mechanism of action studies were performed by the Thibodeau and Brodsky groups using Core supported assays, methodologies and reagents. 

Apical CFTR/ENaC trafficking and activity (Butterworth, Carattino, Frizzell, Kleyman, Hughey, Myerburg, Pilewski, Swiatecka-Urban, Thibodeau): This group focuses on the apical plasma membrane domain, on CFTR and ENaC trafficking, and the protein interactions that determine apical channel density, internalization and apical recycling vs. degradation.  For example, their work has elucidated the regulation of apical ENaC activity by proteases and has determined high intracellular sodium concentration inhibits ENaC subunit maturation during biogenesis.  Regulatory processes and protein interactions that determine apical CFTR and ENaC channel number and activity are therapeutic targets in CF disease. Notably, the secretion of proteases by bacteria, such as P. aeruginosa and M. abscessus, which inhabit CF airways, were shown by this group to contribute to excess Na absorption and reduced ASL volume, providing a rationale for protease inhibitors as therapeutics. Despite these publications appearing during the prior P30 period, there is continued interest in this work, reflected by an invitation to Dr. Thibodeau from the 2017 North American CF Conference to deliver a symposium talk on this topic. This group benefits from the assays and materials (proteins, viral vectors, and functional assays) provided by Core B, from the generation of primary cultures by Core A, which encourage collaborations with outside academic labs and bacterial cloning (Core C).

ENaC polymorphisms modify F508del disease severity (Kleyman, Sheng): Homozygous F508del CF patients who survive longer than predicted for this genotype, with minimal lung function impairment over more than 20 years, underwent whole genome sequencing.  Four of the five individuals carried extremely rare or never reported variants in the SCNN1D and SCNN1B genes of ENaC (channel subunits delta and beta). An enriched rare variant in SCNN1D was identified in the exome variant server database associated with a milder pulmonary disease phenotype. Functional analyses by the Kleyman group revealed two of the three variants encoding delta-ENaC exhibited hypomorphic channel activity. These results suggest a potential role of delta ENaC in control of sodium reabsorption in the airways, and underscore the potential for ENaC as a therapeutic target in CF.