Cystic fibrosis (CF) is the most common lethal genetic disease in the Caucasian population. It is caused by mutations in the CF gene, encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel that belongs to the ATP-binding cassette family. A large number of mutations, including the most common deletion of phenylalalnine 508 (deltaF508) cause the misfolding and intracellular processing defect of CFTR. A functional high-throughput screening (HTS) assay, implemented by Dr. Verkman, has already identified small molecules that partially correct the processing defect of deltaF508 CFTR in Fisher Rat Thyroid (FRT) epithelia and baby hamster kidney (BHK) cells. Since the selection of lead compounds is based on functional criteria, one of the objectives of the CFTR Biochemistry Core is to validate additional primary hits by biochemical methods. This will be achieved by a combination of immunoblotting and metabolic pulse chase technique in conjunction with immunoprecipitation, glycosidase digestion, cell surface biotinylation and antibody binding assays, performed on various epithelia, non-polarized and primary cells. The second major objective of the Core is to elucidate the underlying cellular and biochemical mechanism responsible for correcting the processing defect of the deltaF508 CFTR by small molecules. Considering that both folding defect at the endoplasmic reticulum (ER) and diminished stability at the cell surface contribute to impaired accumulation of deltaF508 CFTR at the plasma membrane, in vivo and in vitro assays will be utilized to unravel mechanistic aspects of the rescue process by small molecules. Verification of the involvement of multiple cellular processes in deltaF508 CFTR rescue (e.g. facilitation of folding at the ER, inhibition of degradation at post-Golgi compartments by promoting recycling and/or deubiquitination and inhibiting internalization) may allow a combination of rescue strategies that act in synergy. The results will provide crucial data in selecting corrector compounds for further development as drugs to treat CF.
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