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.
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).
|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; Rab, Andras; Fu, Lianwu et al. (2011) CFTR expression regulation by the unfolded protein response. Methods Enzymol 491:3-24|
|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|
|Collawn, James F; Fu, Lianwu; Bebok, Zsuzsa (2010) Targets for cystic fibrosis therapy: proteomic analysis and correction of mutant cystic fibrosis transmembrane conductance regulator. Expert Rev Proteomics 7:495-506|
|Jurkuvenaite, Asta; Chen, Lan; Bartoszewski, Rafal et al. (2010) Functional stability of rescued delta F508 cystic fibrosis transmembrane conductance regulator in airway epithelial cells. Am J Respir Cell Mol Biol 42:363-72|
|Bartoszewski, Rafal A; Jablonsky, Michael; Bartoszewska, Sylwia et al. (2010) A synonymous single nucleotide polymorphism in DeltaF508 CFTR alters the secondary structure of the mRNA and the expression of the mutant protein. J Biol Chem 285:28741-8|
|Bartoszewski, Rafal; Rab, Andras; Jurkuvenaite, Asta et al. (2008) Activation of the unfolded protein response by deltaF508 CFTR. Am J Respir Cell Mol Biol 39:448-57|
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