Reprogramming the cells secretory machinery: A cystic fibrosis rescue

Abstract

Cystic fibrosis (CF) is a genetic disorder resulting in the secretion of highly viscous mucus in the airways, leading to lung infection, respiratory failure, morbidity and mortality. CF is attributed to mutation in the CF Transmembrane Conductance Regulator (CFTR) gene that codes for a chloride transporting channel at the cell plasma membrane. More than 2,000 mutations in the CF gene have been identified; however, the {Delta}F508 CFTR is the most common, accounting for approximately 70% of all CFTR mutations. Our earlier studies demonstrate the CFTR protein to be among the nearly 30 proteins constituting the porosome secretory machinery at the plasma membrane in human airway epithelial mucous-secreting cells. Additionally, our recent studies show that stimulated human airway epithelial cells pre-exposed to CFTR inhibitors result in loss of mucus secretion, suggesting the involvement of CFTR in porosome-mediated mucus secretion. To further test the hypothesis that CFTR is involved in porosome-mediated mucus secretion in the human airways, and to develop a therapeutic approach to overcome this defect in {Delta}F508 CFTR human bronchial epithelial cells, the current study was undertaken. Mass spectrometry and Western Blot analysis of porosomes isolated from WT-CFTR Human Bronchial Epithelial (HBE) Cells and {Delta}F508-CFTR CF HBE cells, demonstrate a varying loss or gain of several porosome proteins, including a decrease in the t-SNARE protein SNAP-23 and undetectable levels of the Ras GTPase activating like-protein IQGAP1 in the {Delta}F508-CFTR CF cells. This suggested that mutation in porosome-associated CFTR protein additionally affects other proteins within the porosome secretory machinery, negatively impacting mucus secretion. Hence, to ameliorate defects in mucus secretion in CF, the reconstitution of functional porosomes obtained from WT-CFTR HBE cells into the plasma membrane of {Delta}F508-CFTR mutant cells was performed. Results from the study demonstrate that porosome reconstitution rescues mucus secretion approximately four-fold more effectively than the currently available CF drugs Tezacaftor and Ivacaftor.