Chaperoning the maturation of the cystic fibrosis transmembrane conductance regulator
2001; American Physical Society; Volume: 281; Issue: 1 Linguagem: Inglês
10.1152/ajplung.2001.281.1.l39
ISSN1522-1504
Autores Tópico(s)Cellular transport and secretion
ResumoEDITORIAL FOCUSChaperoning the maturation of the cystic fibrosis transmembrane conductance regulatorJeffrey L. BrodskyJeffrey L. Brodsky Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260Published Online:01 Jul 2001https://doi.org/10.1152/ajplung.2001.281.1.L39MoreSectionsPDF (93 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat the endoplasmic reticulum (ER) serves as a way station during the biogenesis of nearly every integral membrane and secreted protein synthesized in eukaryotic cells. As such, its primary role is to facilitate the folding of nascent polypeptides. Thus plentiful among the ER resident proteins are 1) enzymes that catalyze conformational rearrangements, such as protein disulfide isomerase and peptidylprolyl isomerase; 2) enzymes that chemically modify the nascent polypeptide, such as signal sequence peptidase and oligosaccharyl transferase; and 3) molecules that retain the polypeptide in a soluble, aggregation-free state. The factors constituting this last group are molecular chaperones, many of which were first identified as heat shock proteins (HSPs) because their synthesis is induced when cells are exposed to high temperatures or other stresses. However, most HSPs have cellular homologs that are constitutively produced, and these are named heat shock cognate proteins (HSCs). HSC and HSP molecular chaperones are defined by their molecular masses (in kDa), and a plentiful class found in every cell type are those with a molecular mass of ∼70 kDa (i.e., HSC70 and HSP70).HSP70 and HSC70 hydrolyze ATP concomitant with their binding to and release from polypeptide substrates, and their ATPase activities may be activated by another group of chaperones (21, 33) known as HSP40 or DnaJ homologs. In some cases, the HSP40 cochaperone delivers a polypeptide substrate to the HSP70 or HSC70 (17, 37). With the use of a variety of in vitro techniques, the HSC70 and HSP70 chaperones have been shown to bind preferentially to short stretches of amino acids with overall hydrophobic character (3, 13,31). Such motifs are normally buried within the core of a native protein but may become solvent accessible during protein folding and upon protein denaturation. Thus HSC70 and HSP70 chaperones are ideally suited to aid unfolded proteins en route to their native conformations by limiting protein aggregation.Deletion of the phenylalanine at position 508 in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is the most common mutation associated with CF (27). CFTR most likely functions as a chloride channel in the plasma membrane of airway and other epithelial cells, but cells containing only the ΔF508 variant either lack or harbor variable amounts of the protein at the plasma membrane (7, 16). Both wild-type and the ΔF508 mutant form of CFTR are translocated into the ER membrane, at which time protein folding should commence. However, in vitro studies by Qu and colleagues (25, 26) suggest that the efficiency of ΔF508 CFTR folding is severely impaired compared with that of the wild-type protein. It has also been shown that the ΔF508 mutant protein is unable to undergo an ATP-dependent conformational change in the ER (20, 40). Consistent with an inability of ΔF508 CFTR to fold efficiently, incubation of cells expressing the mutant at lower temperatures, a condition that may favor protein folding and/or stability, permits some of this variant to traffic to the plasma membrane where it is active (10), albeit with a reduced open probability and half-life (9, 11, 19). In contrast, if CFTR folding is retarded in the ER, the protein becomes a substrate for ER-associated degradation, a quality control mechanism that degrades aberrant proteins in the secretory pathway (4). Degradation of CFTR, like most other ER-associated degradation substrates, requires ubiquitinylation and delivery to the multicatalytic cytoplasmic proteasome (14, 36, 38). Although a significant proportion of even wild-type CFTR is degraded, nearly all of the ΔF508 mutant is destroyed; therefore, cells expressing only the mutant form of the protein are phenotypically null.Because ΔF508 CFTR is unable to attain a native conformation in the ER, it should not come as a surprise that it interacts with molecular chaperones. Specifically, cytoplasmic HSC70 and HSP90, the luminal Ca2+-binding chaperone calnexin, and a cytoplasmic HSP40 homolog Hdj2 can be coprecipitated with CFTR (18, 22, 24,39). HSC70 and Hdj2 also prevent the in vitro aggregation of the first nucleotide-binding domain of CFTR (22, 34), the domain in which the phenylalanine at position 508 resides. Although wild-type, folded CFTR is released from the chaperones on its transit from the ER to the Golgi apparatus, HSC70 and calnexin remain bound to the mutant until degradation ensues (24, 39). A HSC70-interacting factor, CHIP, has recently been shown to facilitate the degradation of immature forms of CFTR (23). One interpretation of these combined data is that the chaperone-CFTR complexes represent folding intermediates.Because several human diseases arise from defects in protein folding (4, 35), because of the devastating effects and widespread occurrence of CF, and because a profound amount of information is available on the mechanisms of molecular chaperones, the modulation of protein folding and chaperone activity has been attempted with small molecules and genetic manipulations. For example, it was demonstrated by the Welch (5) and Kopito (32) laboratories that ΔF508 CFTR-expressing cells incubated in glycerol, a polyol known to stabilize proteins in vitro, contain ER-matured forms of CFTR and display cAMP (forskolin)-activated plasma membrane chloride transport, a hallmark of functional CFTR. Glycerol also facilitates the in vitro folding of the first nucleotide-binding domain lacking the phenylalanine at position 508 by preventing off-pathway intermediates from forming (25). ΔF508 CFTR-expressing cells incubated in trimethylamine oxide (TMAO), an osmotic stabilizer found in sharks to protect proteins from urea denaturation, exhibit similar properties (5). Compounds such as TMAO and glycerol are now commonly known as chemical chaperones.Another means to alter protein folding in vivo is through the modulation of intracellular chaperone concentration. For example, Rubenstein et al. (28) showed that sodium 4-phenylbutyrate (4PBA), an ammonia scavenger approved for the treatment of urea cycle disorders and a known transcriptional regulator, permits a fraction of ΔF508 CFTR to mature in bronchial epithelial cell lines and primary nasal epithelial cells from patients with CF. Maturation of the mutant coincides with cAMP-stimulated plasma membrane Cl− conductance in the treated cells. To determine the molecular basis of 4PBA-mediated rescue of the ΔF508 CFTR phenotype, immunoprecipitations were performed to examine the amount of CFTR associated with HSC70, and a 4PBA dose-dependent decrease in the amount of CFTR-HSC70 complex was observed (30). Because HSC70 was shown to facilitate ubiquitination of several test proteins in vitro (2), the authors suggested that HSC70 targets CFTR for degradation and that 4PBA, through an unknown mechanism, reduces the amount of the complex by which CFTR is delivered to the proteasome.The studies described above support the concept of using chaperone modulation as a means to rectify the pathophysiology associated with ΔF508 CFTR. Now, three papers in this issue of the American Journal of Physiology-Lung Cellular and Molecular Physiology(8, 12, 29) represent a significant leap in our understanding of the molecular basis of chaperone modulation, with the ultimate hope that such interventions may become clinically relevant for CF.Building on previous studies, Rubenstein and Lyons (29) show that the mechanism by which 4PBA downregulates HSC70 levels is through a decrease in the stability of HSC70 mRNA. Although HSC70 mRNA is known to be relatively unstable, 4PBA appears to further accelerate its degradation. Coincident with an acceleration of mRNA degradation, an ∼40% reduction in the amount of HSC70 is evident. The mechanism by which this occurs is unknown, but it has been shown that inhibiting the initiation of yeast HSC70 translation leads to a rapid destruction of its message (1). Because a large number of translation poisons are known, many of which were first isolated as antibiotics, this hypothesis can be tested.Does 4PBA only modulate HSC70 levels? Although previous data (30) suggested that this was so, new work from Choo-Kang and Zeitlin (8) indicates that 4PBA raises HSP70 levels, leading to increased HSP70-ΔF508 CFTR complex formation and ΔF508 CFTR maturation. One concern is that this phenomenon may arise from a secondary effect of decreasing HSC70 levels (see above), which, in turn, could induce a general heat shock or stress response. However, the authors show that CFTR maturation is also facilitated by selectively increasing the intracellular levels of HSP70 by transient transfection with an HSP70 expression vector.These combined studies suggest that modulating the levels and thus the activities of HSC70 and HSP70 enhances CFTR maturation. They also point to the complexity of HSP70 or HSC70 function in the cell. Whereas one isoform (i.e., HSP70) facilitates protein folding, its homolog (i.e., HSC70) targets a misfolded protein for degradation. Previous data suggest that multiple chaperones bind coordinately to nascent polypeptides. Although the functions of many of these chaperones are likely to be redundant, it seems logical that a subset is required for folding and another subset is necessary for protein degradation. If one group fails to fold the polypeptide, another is readily available to target the substrate for destruction. The activities of the unique sets of chaperones may be further defined by the action of the HSP40/DnaJ cochaperones that regulate HSP70 and HSC70 function. In other cases, the folding and degradation targeting activities may reside in one chaperone complex. For example, HSP90 has been proposed to facilitate both CFTR maturation and degradation (18).Another means to catalyze CFTR folding is through the use of chemical chaperones, an attack that has shown promise in in vitro model systems as described above. To determine whether this approach is feasible in an animal system, Fischer et al. (12) examined whether TMAO corrects the ΔF508 CFTR trafficking defect in mice. Because the severity of the defect associated with the ΔF508 form of CFTR is most prevalent in the rodent intestine, measurements of rectal potential difference (RPD) were undertaken in control mice, those lacking CFTR, and ΔF508 CFTR mice. Administration of TMAO over 24 h by subcutaneous injection reveals significant increases in forskolin-activated RPD hyperpolarization in the control and ΔF508 CFTR mice, consistent with a partial rescue of the folding defects associated with the wild-type and mutant proteins. Most promising from a clinical perspective is the observation that administering the flavinoid apigenin, a CFTR activator, further increases the forskolin-activated RPD in the ΔF508 CFTR-expressing mouse. This result supports the promise that combined therapeutic attacks may present the best option to lessen the pathophysiology of CF. Combining small-molecule activators of CFTR function is warranted, particularly in this case, because the concentrations of TMAO used for these studies are quite high and the physiological effects of prolonged administration of TMAO are unknown.Might chaperone-based therapies become clinically relevant and generalized to the spectrum of diseases that arise from protein misfolding? The corrective effects of glycerol and 4-PBA on the maturation and secretion of another "conformational disease"-causing protein, the Z variant of antitrypsin, have been recently reported by Burrows et al. (6). In addition, other chaperone modulators are currently being examined for their ability to correct misfolded proteins. 15-Deoxyspergualin, a known HSC70 effector and immunosuppressant, increases the cAMP-stimulated plasma membrane Cl− channel activity of ΔF508 CFTR-expressing cells (15). However, because an increase in ER-matured forms of ΔF508 CFTR could not be observed with 15-deoxyspergualin treatment, only a small amount of the mutant had likely trafficked to the plasma membrane and yielded the desired activity. Nevertheless, with the advent of combinatorial chemistry and more defined methods to examine protein folding and chaperone action at the molecular level, I suspect that we will see a new convergence of chemical, cell biological, and biophysical tools geared to combat a variety of human diseases.FOOTNOTESAddress for reprint requests and other correspondence: J. L. 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Chemical Modulators and Therapeutics1 July 2015Biosynthesis of cystic fibrosis transmembrane conductance regulatorThe International Journal of Biochemistry & Cell Biology, Vol. 52Calpain Inhibition Promotes the Rescue of F508del-CFTR in PBMC from Cystic Fibrosis Patients13 June 2013 | PLoS ONE, Vol. 8, No. 6A Soluble Sulfogalactosyl Ceramide Mimic Promotes ΔF508 CFTR Escape from Endoplasmic Reticulum Associated DegradationChemistry & Biology, Vol. 16, No. 4Entry into the Endoplasmic Reticulum: Protein Translocation, Folding and Quality ControlChaperone displacement from mutant cystic fibrosis transmembrane conductance regulator restores its function in human airway epitheliaThe FASEB Journal, Vol. 22, No. 9The medium is the message: Glycosphingolipids and their soluble analoguesBiochimica et Biophysica Acta (BBA) - General Subjects, Vol. 1780, No. 3The ΔF508 cystic fibrosis mutation impairs domain-domain interactions and arrests post-translational folding of CFTR26 December 2004 | Nature Structural & Molecular Biology, Vol. 12, No. 1Small Molecule Modulators of Endogenous and Co-chaperone-stimulated Hsp70 ATPase ActivityJournal of Biological Chemistry, Vol. 279, No. 49COPII-dependent export of cystic fibrosis transmembrane conductance regulator from the ER uses a di-acidic exit code11 October 2004 | Journal of Cell Biology, Vol. 167, No. 1Evolving questions and paradigm shifts in endoplasmic-reticulum-associated degradation (ERAD)18 August 2003 | BioEssays, Vol. 25, No. 9Disease genes: flattery and deceptionTrends in Pharmacological Sciences, Vol. 23, No. 11CFTR trafficking and signaling in respiratory epitheliumBruce R. Pitt1 July 2001 | American Journal of Physiology-Lung Cellular and Molecular Physiology, Vol. 281, No. 1Recognition and Delivery of ERAD Substrates to the Proteasome and Alternative Paths for Cell Survival More from this issue > Volume 281Issue 1July 2001Pages L39-L42 Copyright & PermissionsCopyright © 2001 the American Physiological Societyhttps://doi.org/10.1152/ajplung.2001.281.1.L39PubMed11404243History Published online 1 July 2001 Published in print 1 July 2001 Metrics Downloaded 233 times
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