Glycerol Reverses the Misfolding Phenotype of the Most Common Cystic Fibrosis Mutation

Abstract

The common ⌬F508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) interferes with the biosynthetic folding of nascent CFTR polypeptides, leading to their retention and rapid degradation in an intracellular compartment proximal to the Golgi apparatus. Neither the pathway by which wild-type CFTR folds nor the mechanism by which the Phe 508 deletion interferes with this process is well understood. We have investigated the effect of glycerol, a polyhydric alcohol known to stabilize protein conformation, on the folding of CFTR and ⌬F508 in vivo. Incubation of transient and stable ⌬F508 tranfectants with 10% glycerol induced a significant accumulation of ⌬F508 protein bearing complex N-linked oligosaccharides, indicative of their transit to a compartment distal to the endoplasmic reticulum (ER). This accumulation was accompanied by an increase in mean whole cell cAMP activated chloride conductance, suggesting that the glycerol-rescued ⌬F508 polypeptides form functional plasma membrane CFTR channels. These effects were dose-and time-dependent and fully reversible. Glycerol treatment also stabilized immature (core-glycosylated) ⌬F508 and CFTR molecules that are normally degraded rapidly. These effects of glycerol were not due to a general disruption of ER quality control processes but appeared to correlate with the degree of temperature sensitivity of specific CFTR mutations. These data suggest a model in which glycerol serves to stabilize an otherwise unstable intermediate in CFTR biosynthesis, maintaining it in a conformation that is competent for folding and subsequent release from the ER quality control apparatus.Cystic fibrosis (CF), 1 a lethal hereditary exocrinopathy affecting approximately one in two thousand live births among populations of Caucasian or northern European descent, is caused by the functional absence of a plasma membrane chloride channel, designated cystic fibrosis transmembrane conductance regulator (CFTR) (1). The vast majority of severe CF cases in these populations is linked to a single genetic lesion, deletion of a phenylalanine codon (⌬F508) (2, 3), which interferes with the folding of newly synthesized CFTR polypeptides. Nascent ⌬F508 molecules fail to traffic to the plasma membrane (4) but rather are retained by the ER quality control mechanism that prevents unfolded or misfolded proteins and unassociated subunits from exiting the ER. Instead, these retained immature ⌬F508 molecules are rapidly degraded (5, 6) in a pre-Golgi compartment by a process that appears to require covalent modification by ubiquitin (7). Moreover, plasma membrane CFTR-like Cl Ϫ channel activity can be detected when ⌬F508 is overexpressed (8) or synthesized at reduced temperature (9), suggesting that Phe 508 does not play an essential role in CFTR function and raising the possibility of therapeutic intervention in CF by increasing the efficiency of ⌬F508 folding.Glycerol and other polyols are known to stabilize protein conformation (10), increase the rate of in vitro protein refolding (11)...

Role of Conserved Prolines in the Structure and Function of the Na+/Dicarboxylate Cotransporter 1, NaDC1

“…We tested both these hypothesis by using chemical chaperones and by constructing double mutants. Chemical chaperones such as glycerol, dimethyl sulfoxide, trimethylamine N-oxide, and 4-phenylbutyric acid have been used to prevent mislocalization of CFTRΔF508 (33), mutants of the aquaporin-2 channel (23) and NaCl cotransporter mutants (34). In the present study, the expression and activity of the P351A and P351G mutants could be restored after treatment with 0.5 M glycerol.…”

Cytoplasmic Loop Three of Cystic Fibrosis Transmembrane Conductance Regulator Contributes to Regulation of Chloride Channel Activity

“…However, maturation of ⌬Phe-508 is sensitive to the cell culture conditions; when cells were grown at reduced temperatures (Denning et al, 1992) or exposed to elevated levels of glycerol (Sato et al, 1996;Brown et al, 1996), an increase of mature ⌬Phe-508 CFTR at the cell surface has been observed. To explore the temperature/glycerol sensitivity of the processing mutants in CL3, cells expressing these mutants were shifted to 26°C for 48 h (Fig.…”

The Proteasome Is Involved in the Degradation of Different Aquaporin-2 Mutants Causing Nephrogenic Diabetes Insipidus

“…Therefore, such differences seem to occur regardless if these are naturally existing glycosylated and nonglycosylated forms of the same protein (our study) or experimentally generated ones. 32 Our observations may be also of relevance for strategies to induce cell surface expression of ER-retained misfolded but functional glycoproteins in which chemical chaperones, 21,33,34 calcium pump inhibitors 35 and low-molecular weight drugs and compounds 36 -38 have been applied to correct the traffic defect. In the case of the T126M AQP2, strategies to preferentially target the glycosylated form should be envisaged.…”