Cystic fibrosis (CF) is the most frequent recessive, hereditary disease in Caucasians. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a chloride channel. CFTR is expressed in the apical membrane of epithelial cells and regulates salt and water homeostasis. The most common form of CF is caused by deletion of phenylalanine at position 508 (?F508). The mutant protein is misfolded and degraded by the ER-associated degradative (ERAD) machinery. Because of its high degree of incidence, ?F508 CFTR has become a popular model of protein folding diseases. ?F508 CFTR is temperature-sensitive and it can be rescued to the cell surface at 27?C or by chemical chaperones, where it retains some activity. Three defects have been identified for ?F508 CFTR: 1) it fails to fold properly and exit the ER;2) the rescued protein is unstable at the cell surface 3) the chloride channel properties are altered. While thousands of published papers have described various aspects of CFTR (and ?F508 CFTR) trafficking and function, virtually all of these employed non-physiological expression contexts such as fibroblast cell lines. In contrast, the fundamental principle guiding our studies is that the choice of model is critical with regard to understanding both WT and ?F508 CFTR biology. We demonstrate that trafficking of the WT or ?F508 CFTR in airway epithelial cells is NOT faithfully recapitulated in cells that that do not express CFTR naturally. We show that ?F508 CFTR is rapidly internalized in polarized airway epithelial cells, whereas wild type (WT) CFTR is not. Our hypothesis is that WT CFTR is normally stable at the apical surface through interactions with a protein complex associated with the cytoskeleton and that ?F508 CFTR has lost this interaction. We therefore propose to study key aspects of WT and ?F508 CFTR biology in polarized epithelial cells. We wish to quantify ?F508 trafficking defects, and understand the mechanisms by which the mutant is destabilized at the plasma membrane. Based on our results that previously selected chemical chaperons are inefficient in rescuing ?F508 CFTR in airway epithelial cells, we identified novel correctors. We are evaluating these molecules in relevant models such as airway epithelial cells, the Cftr?F508 mouse, and primary human bronchial epithelial cells.
The specific aims are:
Aim 1 : To test the hypothesis that the cell surface trafficking differences between WT and ?F508 CFTR result from their alternative association with adaptor complexes and functionally significant binding partners. These differences are the result of ?F508 CFTR misfolding and ubiquitin-dependent degradation;
and Aim 2 : To determine the mechanisms by which novel chemical chaperones rescue and correct ?F508 CFTR. Our goal is to understand how the molecular machinery recognizes aberrant proteins at the plasma membrane. The results of this study are relevant in the analysis of other protein folding diseases.

Public Health Relevance

Protein folding defects are responsible for a large number of human diseases including cystic fibrosis. CFTR, the protein defective in cystic fibrosis, is an excellent model to investigate whether correction of the folding defect will lead to proper function. In the proposed studies, we are examining the cell surface stability of this chloride channel and the cellular mechanisms that regulate its cell surface stability.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK060065-09
Application #
8266404
Study Section
Special Emphasis Panel (ZRG1-CB-N (03))
Program Officer
Mckeon, Catherine T
Project Start
2001-07-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
9
Fiscal Year
2012
Total Cost
$312,297
Indirect Cost
$99,125
Name
University of Alabama Birmingham
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Madanecki, Piotr; Nozell, Susan; Ochocka, Renata et al. (2014) RNAdigest: a web-based tool for the analysis and prediction of structure-specific RNAse digestion results. PLoS One 9:e96759
Collawn, James F; Fu, Lianwu; Bartoszewski, Rafal et al. (2014) Rescuing ?F508 CFTR with trimethylangelicin, a dual-acting corrector and potentiator. Am J Physiol Lung Cell Mol Physiol 307:L431-4
Madanecki, Piotr; Kapoor, Niren; Bebok, Zsuzsa et al. (2013) Regulation of angiogenesis by hypoxia: the role of microRNA. Cell Mol Biol Lett 18:47-57
Londino, James D; Lazrak, Ahmed; Jurkuvenaite, Asta et al. (2013) Influenza matrix protein 2 alters CFTR expression and function through its ion channel activity. Am J Physiol Lung Cell Mol Physiol 304:L582-92
Lazrak, Ahmed; Fu, Lianwu; Bali, Vedrana et al. (2013) The silent codon change I507-ATC->ATT contributes to the severity of the ?F508 CFTR channel dysfunction. FASEB J 27:4630-45
Bartoszewska, Sylwia; Kochan, Kinga; Madanecki, Piotr et al. (2013) Regulation of the unfolded protein response by microRNAs. Cell Mol Biol Lett 18:555-78
Rab, Andras; Rowe, Steven M; Raju, S Vamsee et al. (2013) Cigarette smoke and CFTR: implications in the pathogenesis of COPD. Am J Physiol Lung Cell Mol Physiol 305:L530-41
Collawn, James F; Lazrak, Ahmed; Bebok, Zsuzsa et al. (2012) The CFTR and ENaC debate: how important is ENaC in CF lung disease? Am J Physiol Lung Cell Mol Physiol 302:L1141-6
Fu, Lianwu; Rab, Andras; Tang, Li Ping et al. (2012) Dab2 is a key regulator of endocytosis and post-endocytic trafficking of the cystic fibrosis transmembrane conductance regulator. Biochem J 441:633-43
Lazrak, Ahmed; Jurkuvenaite, Asta; Chen, Lan et al. (2011) Enhancement of alveolar epithelial sodium channel activity with decreased cystic fibrosis transmembrane conductance regulator expression in mouse lung. Am J Physiol Lung Cell Mol Physiol 301:L557-67

Showing the most recent 10 out of 27 publications