scholarly journals Assembly and Misassembly of Cystic Fibrosis Transmembrane Conductance Regulator: Folding Defects Caused by Deletion of F508 Occur Before and After the Calnexin-dependent Association of Membrane Spanning Domain (MSD) 1 and MSD2

2008 ◽  
Vol 19 (11) ◽  
pp. 4570-4579 ◽  
Author(s):  
Meredith F. N. Rosser ◽  
Diane E. Grove ◽  
Liling Chen ◽  
Douglas M. Cyr
Keyword(s):  
Cystic Fibrosis ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Ubiquitin Ligase Complex ◽  
Before And After ◽  
Membrane Spanning ◽  
Polytopic Membrane Protein ◽  
Cystic Fibrosis Transmembrane ◽  
Membrane Spanning Domains

Cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic membrane protein that functions as a Cl− channel and consists of two membrane spanning domains (MSDs), two cytosolic nucleotide binding domains (NBDs), and a cytosolic regulatory domain. Cytosolic 70-kDa heat shock protein (Hsp70), and endoplasmic reticulum-localized calnexin are chaperones that facilitate CFTR biogenesis. Hsp70 functions in both the cotranslational folding and posttranslational degradation of CFTR. Yet, the mechanism for calnexin action in folding and quality control of CFTR is not clear. Investigation of this question revealed that calnexin is not essential for CFTR or CFTRΔF508 degradation. We identified a dependence on calnexin for proper assembly of CFTR's membrane spanning domains. Interestingly, efficient folding of NBD2 was also found to be dependent upon calnexin binding to CFTR. Furthermore, we identified folding defects caused by deletion of F508 that occurred before and after the calnexin-dependent association of MSD1 and MSD2. Early folding defects are evident upon translation of the NBD1 and R-domain and are sensed by the RMA-1 ubiquitin ligase complex.

Biosynthesis and Degradation of CFTR

1999 ◽  
Vol 79 (1) ◽  
pp. S167-S173 ◽  
Author(s):  
RON R. KOPITO
Keyword(s):  
Cystic Fibrosis ◽  
Secretory Pathway ◽  
Covalent Modification ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
The Common ◽  
Effects Of Temperature ◽  
Cystic Fibrosis Transmembrane

Kopito, Ron R. Biosynthesis and Degradation of CFTR. Physiol. Rev. 79, Suppl.: S167–S173, 1999. — Many of the mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that cause cystic fibrosis interfere with the folding and biosynthetic processing of nascent CFTR molecules in the endoplasmic reticulum. Mutations in the cytoplasmic nucleotide binding domains, including the common allele ΔF508, decrease the efficiency of CFTR folding, reduce the probability of its dissociation from molecular chaperones, and largely prevent its maturation through the secretory pathway to the plasma membrane. These mutant CFTR molecules are rapidly degraded by cytoplasmic proteasomes by a process that requires covalent modification by multiubiquitination. The effects of temperature and chemical chaperones on the intracellular processing of mutant CFTR molecules suggest that strategies aimed at increasing the folding yield of this protein in vivo may eventually lead to the development of novel therapies for cystic fibrosis.

scholarly journals Structural comparative modeling of multi-domain ΔF508 CFTR

2021 ◽  
Author(s):  
Eli Fritz McDonald ◽  
Hope Woods ◽  
Shannon Smith ◽  
Minsoo Kim ◽  
Clara T. Schoeder ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Rational Design ◽  
Anion Channel ◽  
Comparative Modeling ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Δf508 Cftr ◽  
Domain Models ◽  
Cystic Fibrosis Transmembrane

Cystic Fibrosis (CF) is a common genetic disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), an epithelial anion channel expressed in several vital organs. Absence of functional CFTR results in imbalanced osmotic equilibrium and subsequent mucus build up in the lungs - which increases the risk of infection and eventually causes death. CFTR is an ATP binding cassette (ABC) transporter composed of two transmembrane domains (TMDs), two nucleotide binding domains (NBDs), and an unstructured regulatory domain. The most prevalent patient mutation is the deletion of F508 (ΔF508), making ΔF508 CFTR the primary target for current FDA approved CF therapies. However, no experimental multi-domain ΔF508 CFTR structure has been determined and few studies have modeled ΔF508 using multi-domain WT CFTR structures. Here, we used cryo-EM density data and Rosetta comparative modeling (RosettaCM) to compare a ΔF508 model with published experimental data on CFTR NBD1 thermodynamics. We then apply this modeling method to generate multi-domain WT and ΔF508 CFTR structural models. These models demonstrate the destabilizing effects of ΔF508 on NBD1 and the NBD1/TMD interface in both the closed and open conformation of CFTR. Furthermore, we modeled ΔF508/R1070W and ΔF508 bound to a the CFTR corrector VX-809. Our models reveal the stabilizing effects of R1070W and VX-809 on multi-domain models of ΔF508 CFTR and pave the way for rational design of additional drugs that target ΔF508 CFTR for treatment of CF.

Cellular ATP release by the cystic fibrosis transmembrane conductance regulator

1996 ◽  
Vol 270 (2) ◽  
pp. C538-C545 ◽  
Author(s):  
A. G. Prat ◽  
I. L. Reisin ◽  
D. A. Ausiello ◽  
H. F. Cantiello
Keyword(s):  
Cystic Fibrosis ◽  
Atp Release ◽  
Functional Expression ◽  
Transmembrane Conductance Regulator ◽  
O2 Consumption ◽  
Transmembrane Conductance ◽  
Intracellular Atp ◽  
Physiological Relevance ◽  
Before And After ◽  
Cystic Fibrosis Transmembrane

Recent studies from our laboratory indicate that members of the ATP-binding cassette (ABC) family of transporters, including P-glycoprotein and cystic fibrosis transmembrane conductance regulator (CFTR), are ATP-permeable channels. The physiological relevance of this novel transport mechanism is largely unknown. In the present study, intra- and extracellular ATP content, cellular ATP release, and O2 consumption before and after adenosine 3',5'-cyclic monophosphate (cAMP) stimulation were determined to assess the role of CFTR in the transport of ATP under physiological conditions. The functional expression of CFTR by the stable transfection of mouse mammary carcinoma cells, C1271, with human epithelial CFTR cDNA resulted in a stimulated metabolism, since both basal and cAMP-inducible O2 consumption were increased compared with mock-transfected cells. The stimulated (but not basal) O2 consumption was inhibited by diphenyl-2-carboxylic acid (DPC), a known inhibitor of CFTR. CFTR expression was also associated with the cAMP-activated and DPC-inhibitable release of intracellular ATP. The recovery of intracellular ATP from complete depletion after metabolic poisoning was also assessed under basal and cAMP-stimulated conditions. The various maneuvers indicate that CFTR may be an important contributor to the release of cellular ATP, which may help modify signal transduction pathways associated with secretory Cl- movement or other related processes. Changes in the CFTR-mediated delivery of nucleotides to the extracellular compartment may play an important role in the onset and reversal of the cystic fibrosis phenotype.

scholarly journals Perturbation of the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Inhibits Its ATPase Activity

2000 ◽  
Vol 276 (15) ◽  
pp. 11575-11581 ◽  
Author(s):  
Ilana Kogan ◽  
Mohabir Ramjeesingh ◽  
Ling-Jun Huan ◽  
Yanchun Wang ◽  
Christine E. Bear
Keyword(s):  
Cystic Fibrosis ◽  
Atpase Activity ◽  
Cross Talk ◽  
Nucleotide Binding ◽  
Cystic Fibrosis Gene ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Cystic Fibrosis Transmembrane

Mutations in the cystic fibrosis gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) lead to altered chloride (Cl−) flux in affected epithelial tissues. CFTR is a Cl−channel that is regulated by phosphorylation, nucleotide binding, and hydrolysis. However, the molecular basis for the functional regulation of wild type and mutant CFTR remains poorly understood. CFTR possesses two nucleotide binding domains, a phosphorylation-dependent regulatory domain, and two transmembrane domains that comprise the pore through which Cl−permeates. Mutations of residues lining the channel pore (e.g.R347D) are typically thought to cause disease by altering the interaction of Cl−with the pore. However, in the present study we show that the R347D mutation and diphenylamine-2-carboxylate (an open pore inhibitor) also inhibit CFTR ATPase activity, revealing a novel mechanism for cross-talk from the pore to the catalytic domains. In both cases, the reduction in ATPase correlates with a decrease in nucleotide turnover rather than affinity. Finally, we demonstrate that glutathione (GSH) inhibits CFTR ATPase and that this inhibition is altered in the CFTR-R347D variant. These findings suggest that cross-talk between the pore and nucleotide binding domains of CFTR may be important in thein vivoregulation of CFTR in health and disease.

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