The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel expressed on the apical surface of epithelial cells and is defective in cystic fibrosis (CF). Our previous project concentrated on the biogenesis of the wild type (WT) and the most common disease causing mutant, ?F508 CFTR. Based on compelling preliminary results, this renewal application focuses on two major topics: 1) the molecular mechanisms by which the unfolded protein response (UPR) regulates gene expression;and 2) the consequences of mRNA secondary structure alterations generated by point mutations using CFTR as our model. We found that the UPR suppresses endogenous WT CFTR transcription, translation, maturation efficiency and function. We have localized the UPR-associated transcriptional repression to the minimal promoter of CFTR. The mechanism of repression involves DNA hypermethylation, histone deacetylation and the binding of ATF6, the UPR-activated transcription factor, to the CFTR minimal promoter. Interestingly, the UPR-associated transcriptional repression affects only a limited number of genes. Regarding the mechanism of decreased CFTR maturation during the UPR, we show that the expression of Hsp90, a pro-folding chaperone of CFTR, is decreased during the UPR. In contrast, the expression of pro-degradation factors increases under the same conditions. Furthermore, the importance of mRNA structure in disease pathogenesis has only recently been recognized. Thus, a very significant finding in our preliminary studies is that the popular model of protein folding disorders, ?F508 CFTR, also exhibits mRNA structural defects. Specifically, the 3- base deletion creates two enlarged hairpin loops in the NBD1 encoding region that slow down translation. This provides the intriguing possibility that F508 CFTR folding, at least to some extent, is compromised because of decreased translation rate caused by mRNA """"""""misfolding"""""""". This aspect of ?F508 CFTR biogenesis has not been examined previously, and has potentially far-reaching implications in other genetic diseases. Our hypotheses are: 1) ER stress activates the UPR which selectively represses the transcription of genes through a promoter-specific mechanism;2) the UPR enhances endoplasmic reticulum-associated degradation (ERAD) by decreasing the expression of pro-folding chaperones and enhancing the level of pro-degradation factors;3) The ?F508 mutation results in mRNA structure defects that decrease translation rate and may promote protein misfolding.
The specific aims are: 1) To identify the mechanisms responsible for transcriptional repression of CFTR during ER stress;2) to understand the mechanisms by which the UPR decreases CFTR maturation efficiency;and 3) to elucidate the role of ?F508 CFTR mRNA secondary structure alterations in protein misfolding and ERAD. These studies will provide novel information regarding the role of the UPR on gene expression regulation and how mRNA structure may contribute to the pathogenesis of genetic disorders.

Public Health Relevance

The goal of the proposed studies is to examine how cellular stress responses affect the expression of a protein that is critical for normal epithelial function, the cystic fibrosis transmembrane conductance regulator, CFTR. Environmental stress such as cigarette smoke causes endoplasmic reticulum stress and activates the unfolded protein response, and the effects of this cellular response on the gene expression profiles of a number of critical genes including CFTR are being examined in these studies. Understanding these processes are important in understanding the cellular basis for a number of lung pathologies including cystic fibrosis and chronic obstructive pulmonary disease (COPD).

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL076587-08
Application #
8241053
Study Section
Cellular and Molecular Biology of the Kidney Study Section (CMBK)
Program Officer
Banks-Schlegel, Susan P
Project Start
2004-05-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2014-03-31
Support Year
8
Fiscal Year
2012
Total Cost
$362,588
Indirect Cost
$115,088
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
Bali, Vedrana; Lazrak, Ahmed; Guroji, Purushotham et al. (2016) A synonymous codon change alters the drug sensitivity of ?F508 cystic fibrosis transmembrane conductance regulator. FASEB J 30:201-13
Bali, Vedrana; Lazrak, Ahmed; Guroji, Purushotham et al. (2016) Mechanistic Approaches to Improve Correction of the Most Common Disease-Causing Mutation in Cystic Fibrosis. PLoS One 11:e0155882
Londino, James David; Lazrak, Ahmed; Noah, James W et al. (2015) Influenza virus M2 targets cystic fibrosis transmembrane conductance regulator for lysosomal degradation during viral infection. FASEB J 29:2712-25
Bali, Vedrana; Bebok, Zsuzsanna (2015) Decoding mechanisms by which silent codon changes influence protein biogenesis and function. Int J Biochem Cell Biol 64:58-74
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
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
Bartoszewski, Rafal; Brewer, Joseph W; Rab, Andras et al. (2011) The unfolded protein response (UPR)-activated transcription factor X-box-binding protein 1 (XBP1) induces microRNA-346 expression that targets the human antigen peptide transporter 1 (TAP1) mRNA and governs immune regulatory genes. J Biol Chem 286:41862-70
Bartoszewski, Rafal; Rab, Andras; Fu, Lianwu et al. (2011) CFTR expression regulation by the unfolded protein response. Methods Enzymol 491:3-24

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