Browsing by Subject "Cystic Fibrosis Transmembrane Conductance Regulator"
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Item The CFTR Folding Pathway: Implications for the Identification and Development of CF Therapeutics(2012-07-20) Mendoza, Juan Luis; Thomas, Philip J.The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is a member of the ABC transporter superfamily, important for Cl- conductance at the apical cell membrane. Loss-of-function of CFTR leads to Cystic Fibrosis (CF), a fatal genetic disease affecting 70,000 people worldwide. There are hundreds of CF causing mutations with the most common being ΔF508, present in at least one allele in 90% of CF patients. CFTR, comprising of 1480 amino acids, folds into five domains important for forming the channel through the membrane, and the regulation of channel function. F508 is located in Nucleotide Binding Domain 1 (NBD1) and is predicted to be at the interface with Intracellular Loop 4 (ICL4) of Transmembrane Domain 2 (TMD2). Studies of the isolated NBD1 demonstrate that the ΔF508 mutation impacts the folding pathway and stability of the domain. Misfolding of NBD1 contributes to the trafficking defect of the intact protein and subsequent loss-of-function. Conversely, second-site suppressor mutations, which more than compensate for defects of the mutant NBD1 domain, only partially rescue CFTR trafficking, suggesting that the deletion also affects other steps along the folding pathway. The aim of this work was to identify positions in CFTR critical for defining the folding pathway. We used a computational approach and two in vitro folding assays to monitor folding of the isolated NBD1 domain and trafficking of full-length CFTR. These data establish a correlation between the folding of the isolated NBD1 domain and maturation of full-length CFTR. Further, NBD1 second-site suppressor mutations in the ΔF508, F508K (NBD1/ICL4 interface disrupting mutation), and R1070W (ΔF508 NBD1/ICL4 interface stabilizing mutation) backgrounds suggest that ΔF508 CFTR is defective in two steps of CFTR biogenesis: 1) stability and efficiency of folding of the NBD1 domain, and 2) NBD1/ICL4 docking. We demonstrate that efficient rescue of ΔF508 CFTR requires correction the two distinct defects. This work has implications for the discovery and development of CF therapeutics by providing a framework for understanding the observed ceiling in the efficacy of either suppressor mutations or corrector compounds, which likely correct a single defect.Item Cotranslational Folding of CFTR(2013-05-31) Patrick, Anna Elizabeth; Thomas, Philip J.; Albanesi, Joseph P.; Gardner, Kevin H.; Jiang, YouxingThe life of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in the cell is dictated by its biogenesis, cellular trafficking, regulated function, and destruction. Cystic fibrosis (CF) is the direct result of perturbations in these processes. Treatment of CF mandates the understanding of the molecular events leading to CFTR loss-of-function. The mechanisms by which different mutations throughout the CFTR protein result in misfolding are unclear. The correction of these processes and ultimately the treatment of CF require elucidation of these mechanisms. In this report, CF-causing mutations in the first transmembrane (TM) spanning domain (TMD1) that result in CFTR misfolding are examined. First, the G85E and G91R mutations in TM1 are shown to have different molecular pathologies. For G85E, TM1 is destabilized in the membrane by the ionizable side chain, which correlates with temperature insensitive ER accumulation. By contrast, G91R does not destabilize TM1, which correlates with temperature sensitive ER accumulation. Both mutants were then identified to perturb TMD1 in a manner recognizable by the cell. Finally, consistent with propagation of these defects, all multidomain CFTR constructs were recognized and degraded in the cell. Other mutations in the interdomain interface between TMD1 and the cytosolic nucleotide binding domains (NBDs) did not perturb TMD1, but affected multidomain constructs containing four domains, which can traffic from the ER. Notably, the interface mutants that change a hydrophobic residue to a basic residue increased levels of early multidomain constructs, suggesting a tradeoff between transient stability and later formation of interdomain interactions. The major cellular monitoring of most mutants occurs after TMD2 is present. In most current models, CF-causing mutations like ?F508 are shown to perturb interdomain interactions before TMD2 is produced. However, evidence presented here suggests these interactions are not important until after TMD2 production. The comparison of TM1 mutants and other mutants supports specific domain interactions in the hierarchical folding model. Taken together, the data herein generate a model of CFTR folding that begins with TMD1. Interdomain interactions then become important in a four domain construct, and the final domain confers additional stability and increases cellular trafficking. Multidomain misfolding clearly plays a role in the molecular pathology of CF, thus, a more detailed understanding of this process as globally outlined above is required to generate novel ways to rescue mutant CFTR.Item Protein Acetylation and Regulation of CFTR Expression(2015-12-28) Vetter, Ali Jean; Chook, Yuh Min; Bruick, Richard K.; De Martino, George; Thomas, Philip J.Cystic Fibrosis (CF) is an autosomal recessive disease caused by a loss of function of a chloride channel encoded by the cystic fibrosis conductance regulator (CFTR) gene. There are several classes of CF-disease causing mutants, the most common lead to protein misfolding, mistrafficking, and retention in the ER before degradation by ERAD. Inhibition of the proteasome does not improve trafficking of the most common misfolded mutant, deltaF508. Several mild mistrafficking mutations were examined under similar conditions to determine if the results with deltaF508 were general or specific. Despite some degree of trafficking, prevention of degradation does not further improve trafficking but instead increases the amount of misfolded protein. Therefore, the decision for retention in the ER is kinetically isolated from proteasome activity. Cotranslational in vitro experiments were implemented to explore the possibility of unidentified proteins interacting with the CFTR nascent chain. Two proteins identified in close proximity to a severe mistrafficking mutant, G85E, were N-alpha-acetyltransferases 16 and 15, both components of the complex NatA. However, the NatB complex, not NatA, is predicted to acetylate the N-terminus of CFTR. Nat complexes can affect the stability of their substrates either through modulating degradation or stability. Knockdown experiments ruled out involvement of NatE, NAA50, as well as one of the auxiliary subunits of NatA, NAA16, in the involvement of CFTR regulation. By contrast, knockdown of both NatA and NatB components increase steady state levels of CFTR. These increases were not due to any changes in degradation rates of the protein or the direct acetylation of CFTR's N-terminus as a Q2P mutation that prevents N-terminal acetylation of all known substrates was without effect. Instead, NatA regulates the translation rate of G85E CFTR through alterations in the mRNA levels. NatB regulates CFTR through a posttranslational maturation step independent of its fitness for proteasomal degradation. Thus, acetylation plays a role in the regulation of CFTR expression through two separate mechanisms, neither involving the direct N-terminal acetylation or degradation of G85E CFTR. Inhibition of either proteasomal or acetylation activity does not restore trafficking of CFTR, but instead increases the misfolded, ER-retained form of the protein.Item Transmembrane Protein Folding: Effects of Disease-Causing Mutations on CFTR Folding and Assembly(2006-05-16) Thibodeau, Patrick Harlan; Thomas, Philip J.The biosyntheses of multi-domain membrane proteins are complex processes which involve the translation, folding, and assembly of domains to reach the native state. The nascent chain of a membrane protein must interact with multiple solvent environments, ribosome and chaperone components, and processing and trafficking machinery, and each of these steps are at least partially determined by the protein sequence and structure. Alterations to protein sequences often perturb these processes by impacting any of a number of structural states of the protein, and while some mutations impact the native state structures of proteins directly, others impact the folding process and have little direct effect on the native state structure. A growing number of mutations have been shown to impact these folding processes in the cystic fibrosis transmembrane conductance regulator (CFTR), a multi-domain transmembrane protein associated with cystic fibrosis. Two such mutations are detailed in this work: F508del and P205S. The most common CF-causing mutation, F508del, is the deletion of a single phenylalanine residue in a cytosolic domain of CFTR and results in a protein which fails to fold at physiological temperature, is retained in the ER and is degraded by the proteasome. The resulting loss of protein is the underlying basis for cystic fibrosis. The loss of the backbone at this position induces the misfolding of the domain, while changes in sidechain character impact subsequent domain-domain assembly. The rescue of full-length F508del CFTR by second-site suppressors correlates with the rescue of the folding of the soluble domain, further suggesting the direct role of Phe508 in domain folding. The mutation of equivalent residues in homologous proteins results in similar phenotypes, suggesting an evolutionary conservation of function for this position. The Pro205 residue, in the first transmembrane domain, has been shown to facilitate proper folding by disfavoring alternate, non-native protein conformations. A computational study of proline residues in transmembrane helices suggests that this mechanism is also conserved evolutionarily. With these data, a hierarchical model for CFTR folding is presented and mechanisms by which these mutations specifically impact the stepwise folding and assembly of CFTR are suggested.