Cystic Fibrosis (CF) is a fatal lung disease characterized by fibrotic tissue damage resultant from defective hydration and chronic bacterial infection of lung airways. The etiology of most CF cases is inheritance of CFTR?F508, a mutant Cl- channel, which is prone to misfolding and premature proteasomal degradation at its site of synthesis in the endoplasmic reticulum (ER). CFTR contains two membrane-spanning domains (MSD), two nucleotide-binding domains (NBD) and a regulatory domain that require proper assembly for channel activity. CFTR?F508is synthesized, but its assembly arrests at an unknown intermediate step and it is selected for degradation prior to passage to the plasma membrane (PM). Assembly defects in CFTR?F508can be rescued by compounds that alter the cellular folding environment and """"""""chemical chaperones"""""""" are being pursued as CF drugs. This competitive renewal application for R01 GM56981 seeks to aid in the development of CF therapeutics by elucidating defective steps in CFTR?F508folding and identifying ER quality control (ERQC) factors that select CFTR?F508for degradation. Preliminary studies identify a novel ER membrane associated E3 ubiquitin ligase complex composed of the ERQC factor Derlin-1, the transmembrane E3 RMA1, the Hsp40 DNAJ12and cytosolic Hsp70 that acts in epithelia to select CFTR for degradation. Expression of RMA1 is strongly induced by inflammatory signals in bacterially infected of CF lungs. Thus, over activity of the RMA1 E3 complex may exacerbate defects in CFTR?F508 biogenesis and because additional defects in protein homeostasis that contribute to the pathology of CF. Thus, we propose to define the mechanism for RMA1 E3 action in the selection of CFTR?F508 for proteasomal degradation. During this process we will determine the portfolio of RMA1 E3 clients that are negatively impacted by chronic over activation of ERQC in inflamed lung epithelial cells. This information will be utilized to develop methods to attenuate RMA1 expression and to rescue CFTR F508 from premature degradation and limit damage to protein homeostasis in epithelia resultant from chronic inflammatory stress. These studies will contribute to development of a treatment for CF and provide a basic understanding of regulation of protein homeostasis during innate immune signaling. !

Public Health Relevance

Cystic Fibrosis, the most common inherited lethal disease among Caucasians, is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The CFTR protein acts as a gated Cl- ion channel at the apical membrane of epithelial cells, thereby facilitating proper hydration of mucosal linings. Disease causing mutations in the CFTR protein can affect a variety of steps in the biogenesis of a functional protein including the folding and trafficking of CFTR as well as the channel activity of plasma membrane-localized protein. The most common mutation in CFTR is the deletion of F508, which leads to the production of a functional channel that has conditional folding defects that lead to its premature degradation. Therefore, current research is focused on the development of approaches to correct folding defects in CFTR?F508. This grant application seeks to define methods to increase CFTR?F508folding efficiency by development of an understanding of the pathway for folding, trafficking and quality control of CFTR. Our approach is carrying out preliminary studies in model cells and translates this work to test models generated in primary airway cells. Our current focus is to extend our preliminary work that identified protein quality control factors that degrade CFTR?F508 and develop methods for correction of an intrinsic folding defect in CFTR F508. In preliminary studies, we developed approaches to restore CFTR?F508 folding to 50% of wild-type levels. CF patients that exhibit around 35% of wild-type CFTR activity have mild disease symptoms, so there is potential for our studies to provide a clinical benefit.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Membrane Biology and Protein Processing (MBPP)
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Wehrle, Janna P
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
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