Open Access Dissertation
Date of Award
Cystic Fibrosis (CF) is the most common, lethal autosomal recessive disorder, and is caused by mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR), an anion channel that is found in most epithelial cells lining the airway and gut. The most common mutation of CFTR is deletion of phenylalanine at position 508 (CFTR-F508del), which produces a misfolded protein. Through the ubiquitin proteasome system (UPS), this misfolded protein is ubiquitinated and signaled for degradation via the cytosolic proteasome. Previous studies demonstrating experimental restoration of F508DEL-CFTR trafficking to the plasma membrane showed partial function of the chloride channel, raising therapeutic speculations. Some components of how F508DEL-CFTR is degraded is known, however many mechanisms that underly its degradation through ERAD and the UPS is still unknown. In the first part of the study we used an siRNA screen to discover the E3 ligase, RNF19B, and its interacting partner, UBE 2L6 mediates F508del degradation. We used siRNA-mediated silencing of endogenous UBE 2L6 and RNF19B in the CF human bronchial epithelial (HBE) cell line to demonstrate that there is an increase in F508del-CFTR expression. We also co-expressed UBE 2L6 and RNF 19B with F508del in HEK293 cells that demonstrated a decrease in F508del compared to control. Cycloheximide-chase (CHX) experiments using HEK 293 cells overexpressing RNF19B and F508del showed that there was a decrease in F508del half-life. Lastly, using siRNA-mediated silencing of endogenous UBE 2L6 and RNF19B in CFBE-F508del cells increased forskolin-stimulated short-circuit currents.
Cell models have given the CF research community a tremendous amount information about the mechanisms that underly the disease. However, they are not suited to understand the pathophysiology of the disease. Existing animal models of CF have limitations because they either fail to exhibit key CF pulmonary phenotypes, die soon after birth, and/or require special care with high maintenance costs. In the second part of the study, we report the generation by CRISPR/Cas9 of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits untreated live for > 40 d, and therapeutic regimens directed towards restoration of intraluminal transit of gastrointestinal materials extend lifespan to > 80 days. CFTR expression in the rabbit lung mimicked expression in the human lung with widespread expression in proximal and distal lower airway epithelia. CF rabbits exhibited human-like abnormalities in airway bioelectric properties and sporadic, spontaneous polymicrobial lower respiratory tract infections in the absence of submucosal glands. The CF rabbit model may serve as a useful tool for understanding CF pathogenesis and the practical development of therapeutics for this life-shortening disease.
Rajagopalan, Carthic, "Molecular Mechanisms In Cftr-F508del Degradation And The Functional Defect Of Cftr Absence In Rabbits" (2019). Wayne State University Dissertations. 2290.