Folding Mutants Research Articles

Active‐site‐specific chaperone therapy for Fabry disease

Protein misfolding is recognized as an important pathophysiological cause of protein deficiency in many genetic disorders. Inherited mutations can disrupt native protein folding, thereby producing proteins with misfolded conformations. These misfolded proteins are consequently retained and degraded by endoplasmic reticulum-associated degradation, although they would otherwise be catalytically fully or partially active. Active-site directed competitive inhibitors are often effective active-site-specific chaperones when they are used at subinhibitory concentrations. Active-site-specific chaperones act as a folding template in the endoplasmic reticulum to facilitate folding of mutant proteins, thereby accelerating their smooth escape from the endoplasmic reticulum-associated degradation to maintain a higher level of residual enzyme activity. In Fabry disease, degradation of mutant lysosomal alpha-galactosidase A caused by a large set of missense mutations was demonstrated to occur within the endoplasmic reticulum-associated degradation as a result of the misfolding of mutant proteins. 1-Deoxygalactonojirimycin is one of the most potent inhibitors of alpha-galactosidase A. It has also been shown to be the most effective active-site-specific chaperone at increasing residual enzyme activity in cultured fibroblasts and lymphoblasts established from Fabry patients with a variety of missense mutations. Oral administration of 1-deoxygalactonojirimycin to transgenic mice expressing human R301Q alpha-galactosidase A yielded higher alpha-galactosidase A activity in major tissues. These results indicate that 1-deoxygalactonojirimycin could be of therapeutic benefit to Fabry patients with a variety of missense mutations, and that the active-site-specific chaperone approach using functional small molecules may be broadly applicable to other lysosomal storage disorders and other protein deficiencies.

Chemical chaperone-mediated protein folding: stabilization of P22 tailspike folding intermediates by glycerol

Polyol co-solvents such as glycerol increase the thermal stability of proteins. This has been explained by preferential hydration favoring the more compact native over the denatured state. Although polyols are also expected to favor aggregation by the same mechanism, they have been found to increase the folding yields of some large, aggregation-prone proteins. We have used the homotrimeric phage P22 tailspike protein to investigate the origin of this effect. The folding of this protein is temperature-sensitive and limited by the stability of monomeric folding intermediates. At non-permissive temperature (>or=35 degrees C), tailspike refolding yields were increased significantly in the presence of 1-4 m glycerol. At low temperature, tailspike refolding is prevented when folding intermediates are destabilized by the addition of urea. Glycerol could offset the urea effect, suggesting that the polyol acts by stabilizing crucial folding intermediates and not by increasing solvent viscosity. The stabilization effect of glycerol on tailspike folding intermediates was confirmed in experiments using a temperature-sensitive folding mutant protein, by fluorescence measurements of subunit folding kinetics, and by temperature up-shift experiments. Our results suggest that the chemical chaperone effect of polyols observed in the folding of large proteins is due to preferential hydration favoring structure formation in folding intermediates.

Observation of sequential steps in the folding of intestinal fatty acid binding protein using a slow folding mutant and19F NMR

The rat intestinal fatty acid binding protein (IFABP) primarily comprises two beta-sheet structures surrounding a large internal cavity. The urea denatured WT protein folds within seconds after dilution to nondenaturing conditions. Replacing a glycine with valine in the turn between the last two beta-strands (Gly121Val) slows the folding process by more than three orders of magnitude. After incorporating 4-(19)F-phenylalanine into the mutant protein, we were able to directly monitor the behavior of the eight phenylalanine side chains in real time during folding using (19)F NMR. Specifically, there is a nonnative-like collapse in regions involving three phenylalanine residues (Phe-62, Phe-68, and Phe-93) within milliseconds. At least two distinct NMR peaks were observed, suggesting conformational fluctuations in this region. Formation of this site is followed by formation of native structure of Phe-2 and Phe-17, then by Phe-47, and finally by the cooperative rearrangement of the intermediate structures to the final native structure. It is proposed that the Gly121Val mutation slows the formation of a normal nucleating site, not only slowing overall folding, but also allowing intermediates in regions distant from the mutation to be experimentally observed. Because intermediates involved in protein folding are normally difficult to observe due to their marginal stability, the experimental approach used here may serve as a general method for determining the nature of both early and late steps in protein folding.

Tuning λ 6–85 Towards Downhill Folding at its Melting Temperature

The five-helix bundle λ 6–85 * is a fast two-state folder. Several stabilized mutants have been reported to fold kinetically near-downhill or downhill. These mutants undergo a transition to two-state folding kinetics when heated. It has been suggested that this transition is caused by increased hydrophobicity at higher temperature. Here we investigate two histidine-containing mutants of λ 6–85 * to see if a weaker hydrophobic core can extend the temperature range of downhill folding. The very stable λHA is the fastest-folding lambda repressor to date ( k f − 1 ≈ k obs − 1 = 2.3 μs at 44 °C). It folds downhill at low temperature, but transits back to two-state folding at its unfolding midpoint. λHG has a weakened hydrophobic core. It is less stable than some slower folding mutants of λ 6–85 *, and it has more exposed hydrophobic surface area in the folded state. This mutant nonetheless folds very rapidly, and has the non-exponential folding kinetics of an incipient downhill folder even at the unfolding midpoint ( k m − 1 ≈ 2 μs, k a − 1 = 15 μs at 56 °C). We also compare the thermodynamic melting transition of λHG with the nominal two-state folding mutant λQG, which has a similar melting temperature. Unlike λQG, λHG yields fluorescence wavelength-dependent cooperativities and probe-dependent melting temperatures. This result combined with previous work shows that the energy landscapes of lambda repressor mutants support all standard folding mechanisms.

Derlin-1 Promotes the Efficient Degradation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and CFTR Folding Mutants

A complex involving Derlin-1 and p97 mediates the retrotranslocation and endoplasmic reticulum (ER)-associated degradation of misfolded proteins in yeast and is used by certain viruses to promote host cell protein degradation (Romisch, K. (2005) Annu. Rev. Cell Dev. Biol. 21, 435-456; Lilley, B. N., and Ploegh, H. L. (2004) Nature 429, 834-840; Ye, Y., Shibata, Y., Yun, C., Ron, D., and Rapoport, T. A. (2004) Nature 429, 841-847). We asked whether the components of this pathway are involved in the endoplasmic reticulum-associated degradation of the mammalian integral membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), a substrate for the ubiquitin-proteasome system. We report that Derlin-1 and p97 formed complexes with CFTR in human airway epithelial cells. Derlin-1 interacted with nonubiquitylated CFTR, whereas p97 associated with ubiquitylated CFTR. Exogenous expression of Derlin-1 led to its co-localization with CFTR in the ER where it reduced wild type (WT) CFTR expression and efficiently degraded the disease-associated CFTR folding mutants, DeltaF508 and G85E (>90%). Consistent with this, Derlin-1 also reduced the amount of WT or DeltaF508 CFTR appearing in detergent-in-soluble aggregates. An approximately 70% knockdown of endogenous Derlin-1 by RNA interference increased the steady-state levels of WT and DeltaF508 CFTR by 10-15-fold, reflecting its significant role in CFTR degradation. Derlin-1 mediated the degradation of N-terminal CFTR fragments corresponding to the first transmembrane domain of CFTR, but CFTR fragments that incorporated additional domains were degraded less efficiently. These findings suggest that Derlin-1 recognizes misfolded, nonubiquitylated CFTR to initiate its dislocation and degradation early in the course of CFTR biogenesis, perhaps by detecting structural instability within the first transmembrane domain.

Why molecular chaperones buffer mutational damage: a case study with a yeast Hsp40/70 system.

The malfunctioning of molecular chaperones may result in uncovering genetic variation. The molecular basis of this phenomenon remains largely unknown. Chaperones rescue proteins unfolded by environmental stresses and therefore they might also help to stabilize mutated proteins and thus mask damages. To test this hypothesis, we carried out a genomewide mutagenesis followed by a screen for mutations that were synthetically harmful when the RAC-Ssb1/2 cytosolic chaperones were inactive. Mutants with such a phenotype were found and mapped to single nucleotide substitutions. However, neither the genes identified nor the nature of genetic lesions implied that folding of the mutated proteins was being supported by the chaperones. In a second screen, we identified temperature-sensitive (ts) mutants, a phenotype indicative of structural instability of proteins. We tested these for an association with sensitivity to loss of chaperone activity but found no such correlation as might have been expected if the chaperones assisted the folding of mutant proteins. Thus, molecular chaperones can mask the negative effects of mutations but the mechanism of such buffering need not be direct. A plausible role of chaperones is to stabilize genetic networks, thus making them more tolerant to malfunctioning of their constituents.

Influence of the antipsychotic drug pipamperone on the expression of the dopamine D4 receptor

The dopamine D4 receptor is a G protein-coupled receptor that binds with high affinity various antipsychotics. The receptor may be involved in attention/cognition, and in genetic studies a polymorphic repeat sequence in its coding sequence has been associated with attention deficit/hyperactivity disorder. We developed an inducible episomal expression system based on the reverse tetracycline transactivator and Epstein-Barr viral sequences. In HEK293rtTA cells expressing the dopamine D4 receptor from this episomal expression vector, addition of doxycycline in combination with sodium butyrate and trichostatin A induces high levels of receptor expression, resulting in 1970 ± 20 fmol/mg membrane protein. Addition of the dopamine D4 receptor and serotonin 5-HT2A receptor antagonist pipamperone to these cells further increased the expression of the dopamine receptor, reaching 3800 ± 60 fmol/mg membrane protein. This up-regulation was not restricted to the dopamine D4 receptor but was also found for the serotonin 5-HT2A receptor. We further provide evidence that the increase in receptor expression is not due to increased mRNA synthesis. As pipamperone could rescue the expression of a folding mutant of the dopamine D4 receptor (M345), we propose that pipamperone acts as a pharmacological chaperone for correct receptor folding thereby resulting in an increased dopamine D4 receptor expression. This study describes a strong and inducible expression system for proteins, difficult to express in other heterologous expression systems. This study also demonstrates that pipamperone, an antipsychotic, acts as a pharmacological chaperone and by doing so, increases the expression level of the dopamine D4 receptor. The fact that ligands can also act as pharmacological chaperones is a fairly new additional element in the regulation of receptor expression levels with potential great impact in drug treatment.

Selective Inhibition of Endoplasmic Reticulum-associated Degradation Rescues ΔF508-Cystic Fibrosis Transmembrane Regulator and Suppresses Interleukin-8 Levels: THERAPEUTIC IMPLICATIONS

Selective Inhibition of Endoplasmic Reticulum-associated Degradation Rescues ΔF508-Cystic Fibrosis Transmembrane Regulator and Suppresses Interleukin-8 Levels: THERAPEUTIC IMPLICATIONS

Specific Rescue of Cystic Fibrosis Transmembrane Conductance Regulator Processing Mutants Using Pharmacological Chaperones

Most mutants of the cystic fibrosis transmembrane conductance regulator (CFTR) that cause severe symptoms of cystic fibrosis do not reach the cell surface because they are defective in folding. Many CFTR folding mutants, however, including the DeltaF508 mutant found in more than 90% of cystic fibrosis patients, are potentially functional at the cell surface if they can be induced to fold correctly. In a previous study (Mol Pharm 2:407-413, 2005), we reported that a quinazoline derivative (CFcor-325) could rescue CFTR processing mutants. The corrector was not specific however, as it could also rescue a processing mutant of CFTR's sister protein, the multidrug resistance P-glycoprotein. The goal of this study was to test whether it was possible to specifically rescue CFTR processing mutants using a pharmacological chaperone. In this article, we report that two compounds, 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol (CFpot-532) and 2-phenylamino-4-(4-ethylene-phenyl)-thiazole (corr-2b) could rescue CFTR processing mutants such as DeltaF508 CFTR but not a P-glycoprotein processing mutant. The compound CFpot-532 also acts as a potentiator of DeltaF508 CFTR channel activity. Therefore, the results suggest that the mechanism whereby CFpot-532 and corr-2b promote folding of CFTR processing mutants is through direct interaction with the CFTR mutant proteins. The compound CFpot-532 could be a particularly useful lead compound for treatment of cystic fibrosis because it is both a CFTR channel potentiator as well as a specific pharmacological chaperone.

Progressive Disruption of Cellular Protein Folding in Models of Polyglutamine Diseases

Numerous human diseases are associated with the chronic expression of misfolded and aggregation-prone proteins. The expansion of polyglutamine residues in unrelated proteins is associated with the early onset of neurodegenerative disease. To understand how the presence of misfolded proteins leads to cellular dysfunction, we employed Caenorhabditis elegans polyglutamine aggregation models. Here, we find that polyglutamine expansions disrupted the global balance of protein folding quality control, resulting in the loss of function of diverse metastable proteins with destabilizing temperature-sensitive mutations. In turn, these proteins, although innocuous under normal physiological conditions, enhanced the aggregation of polyglutamine proteins. Thus, weak folding mutations throughout the genome can function as modifiers of polyglutamine phenotypes and toxicity.

Principal component analysis of fast-folding λ-repressor mutants

Principal component (PC) analysis is carried out on 10 ns simulations of four fast-folding mutants of the λ 6–85-repressor fragment near the native structure. The range of motion along the largest two PCs is seen to be greater for the faster folding mutants in the group. Dynamics along the largest PC and the free energy profile resemble those for Brownian dynamics, the former apparently more than thus far found for other proteins over similar times. These measures, however, do not easily distinguish Brownian from subdiffusive dynamics, which is suggested by the PC eigenvalue distribution.

Mechanisms of Pharmacological Rescue of Trafficking-defective hERG Mutant Channels in Human Long QT Syndrome

Long QT syndrome type 2 is caused by mutations in the human ether-a-go-go-related gene (hERG). We previously reported that the N470D mutation is retained in the endoplasmic reticulum (ER) but can be rescued to the plasma membrane by hERG channel blocker E-4031. The mechanisms of ER retention and how E-4031 rescues the N470D mutant are poorly understood. In this study, we investigated the interaction of hERG channels with the ER chaperone protein calnexin. Using coimmunoprecipitation, we showed that the immature forms of both wild type hERG and N470D associated with calnexin. The association required N-linked glycosylation of hERG channels. Pulse-chase analysis revealed that N470D had a prolonged association with calnexin compared with wild type hERG and E-4031 shortened the time course of calnexin association with N470D. To test whether the prolonged association of N470D with calnexin is due to defective folding of mutant channels, we studied hERG channel folding using the trypsin digestion method. We found that N470D and the immature form of wild type hERG were more sensitive to trypsin digestion than the mature form of wild type hERG. In the presence of E-4031, N470D became more resistant to trypsin even when its ER-to-Golgi transport was blocked by brefeldin A. These results suggest that defective folding of N470D contributes to its prolonged association with calnexin and ER retention and that E-4031 may restore proper folding of the N470D channel leading to its cell surface expression.

Heterologous expression of membrane and soluble proteins derepresses GCN4 mRNA translation in the yeast Saccharomyces cerevisiae.

This paper describes the first physiological response at the translational level towards heterologous protein production in Saccharomyces cerevisiae. In yeast, the phosphorylation of eukaryotic initiation factor 2alpha (eIF-2alpha) by Gcn2p protein kinase mediates derepression of GCN4 mRNA translation. Gcn4p is a transcription factor initially found to be required for transcriptional induction of genes responsible for amino acid or purine biosynthesis. Using various GCN4-lacZ fusions, knockout yeast strains, and anti-eIF-2alpha-P/anti-eIF-2alpha antibodies, we observed that heterologous expression of the membrane-bound alpha1beta1 Na,K-ATPase from pig kidney, the rat pituitary adenylate cyclase seven-transmembrane-domain receptor, or a 401-residue soluble part of the Na,K-ATPase alpha1 subunit derepressed GCN4 mRNA translation up to 70-fold. GCN4 translation was very sensitive to the presence of heterologous protein, as a density of 1 per thousand of heterologous membrane protein derepressed translation maximally. Translational derepression of GCN4 was not triggered by misfolding in the endoplasmic reticulum, as expression of the wild type or temperature-sensitive folding mutants of the Na,K-ATPase increased GCN4 translation to the same extent. In situ activity of the heterologously expressed protein was not required for derepression of GCN4 mRNA translation, as illustrated by the expression of an enzymatically inactive Na,K-ATPase. Two- to threefold overexpression of the highly abundant and plasma membrane-located endogenous H-ATPase also induced GCN4 translation. Derepression of GCN4 translation required phosphorylation of eIF-2alpha, the tRNA binding domain of Gcn2p, and the ribosome-associated proteins Gcn1p and Gcn20p. The increase in Gcn4p density in response to heterologous expression did not induce transcription from the HIS4 promoter, a traditional Gcn4p target.

717. VCP Short Hairpin RNA Rescues ΔF508- CFTR and Suppresses IL8 Levels: Therapeutic Implications in Cystic Fibrosis

Top of pageAbstract Background: Endoplasmic reticulum associated degradation (ERAD) is the major quality control pathway of the cell. The most common disease-causing protein folding mutation, |[Delta]|F508-CFTR, is destroyed by ERAD to cause cystic fibrosis (CF). Valosin containing protein (VCP) is an AAA ATPase that participates in retrograde translocation of misfolded proteins from the ER and degradation of polyubiquitinated protein by the proteasomes. We hypothesize that selective inhibition of VCP not only rescues |[Delta]|F508-CFTR but also suppresses IL8 cytokine levels, the major inflammatory cytokine in CF airways. Methods: The VCP shRNA was PCR amplified from oligo/template containing the VCP hairpin using universal primers which contain XhoI (5' prime) and EcoRI (3' prime) flank. The PCR fragment was then cloned into the hairpin cloning site of pSHAG-MAGIC2/pSM2 vector (Open Biosystems). IB3-1 (|[Delta]|F508/W1282X) bronchial epithelial cells were transiently transfected with VCP and gp78 shRNA/siRNA constructs for selective inhibition of ERAD. CFTR levels and chloride efflux were measured by metabolic labeling and MQAE fluorescence assay, respectively. Proteasome inhibition in IB3-1 cells was carried out using PS-341 (Bortezomib/Velcade) for 6hrs. The IL8 cytokine levels were measured in IL1-|[beta]| induced IB3-1 cells after ERAD or proteasome inhibition. Findings: We show here that p97/VCP and gp78 form complexes with CFTR during translocation from the ER for degradation by the cytosolic proteasome. Interference in the VCP/CFTR complex promotes accumulation of immature CFTR in the ER and partial rescue of functional chloride channels to the cell surface. Moreover, under these conditions, the cytokine whose expression is regulated by the proteasome, IL8, is reduced. Inhibition of the proteasome with bortezomib (PS-341/Velcade) also rescues CFTR and suppresses IL8 levels, but with less efficiency. Interpretation: We propose that selective inhibition of ERAD by VCP short hairpin RNA is superior to gp78 siRNA or proteasome inhibition in the correction of the |[Delta]|F508 CFTR trafficking defect.

A selection for mutants that interfere with folding of Escherichia coli thioredoxin-1 in vivo

Escherichia coli thioredoxin is normally a cytoplasmic protein involved in the reduction of disulfide bonds. However, thioredoxin can be translocated to the periplasm when it is attached to a cotranslational signal sequence. When exported to the periplasm, it can partially replace the activity of DsbA in promoting the formation of disulfide bonds. In contrast, when thioredoxin is fused to a posttranslational signal sequence, very little of it appears in the periplasm. We propose that this absence of posttranslational export is due to the rapid folding of thioredoxin in the cytoplasm. We sought mutants of thioredoxin that retarded its folding in the cytoplasm, which we accomplished by fusing thioredoxin to a posttranslational signal sequence and selecting for mutants in which thioredoxin was exported to the periplasm, where it could replace DsbA. The collection of mutants obtained represents a limited number of amino acid changes in the protein. In vitro studies on purified mutant proteins show that all but one are defective in the kinetics and thermodynamics of protein folding. We propose that the slower folding of the thioredoxin mutant proteins in the cytoplasm allows their export by a posttranslational pathway. We discuss some implications of this class of mutants for aspects of the folding pathway of thioredoxin and for its mechanism of export. In particular, the finding that a folding mutant that allows protein translocation alters an amino acid at the C terminus of the protein suggests that the degree to which thioredoxin folds during its translation must be severely restricted.

Uncoupling of Brefeldin A‐Mediated Coatomer Protein Complex‐I Dissociation from Golgi Redistribution

The Golgi complex functions in transport of molecules from the endoplasmic reticulum (ER) to the plasma membrane and other distal organelles as well as in retrograde transport to the ER. The fungal metabolite brefeldin A (BFA) promotes dissociation of ADP-ribosylation-factor-1 (ARF1) and the coatomer protein complex-I (COP-I) from Golgi membranes, followed by Golgi tubulation and fusion with the ER. Here we demonstrate that the cationic ionophore monensin inhibited the BFA-mediated Golgi redistribution to the ER without interfering with ARF1 and COP-I dissociation. Preservation of a perinuclear Golgi despite COP-I and ARF1 dissociation enables addressing the involvement of these proteins in anterograde ER to Golgi transport. The thermo-reversible folding mutant of vesicular stomatitis virus G protein (VSVGtsO45) was retained in the ER in the presence of both monensin and BFA, thus supporting ARF1/COP-I participation in ER-exit processes. Live-cell imaging revealed that BFA-induced Golgi tubulation persisted longer in the presence of monensin, suggesting that monensin inhibits tubule fusion with the ER. Moreover, monensin also augmented Golgi-derived tubules that contained the ER-Golgi-intermediate compartment marker, p58, in the absence of BFA, signifying the generality of this effect. Taken together, we propose that monensin inhibits membrane fusion processes in the presence or absence of BFA.

Evolutionary and experimental analyses of inorganic phosphate transporter PiT family reveals two related signature sequences harboring highly conserved aspartic acids critical for sodium‐dependent phosphate transport function of human PiT2

The mammalian members of the inorganic phosphate (P(i)) transporter (PiT) family, the type III sodium-dependent phosphate (NaP(i)) transporters PiT1 and PiT2, have been assigned housekeeping P(i) transport functions and are suggested to be involved in chondroblastic and osteoblastic mineralization and ectopic calcification. The PiT family members are conserved throughout all kingdoms and use either sodium (Na+) or proton (H+) gradients to transport P(i). Sequence logo analyses revealed that independent of their cation dependency these proteins harbor conserved signature sequences in their N- and C-terminal ends with the common core consensus sequence GANDVANA. With the exception of 10 proteins from extremophiles all 109 proteins analyzed carry an aspartic acid in one or both of the signature sequences. We changed either of the highly conserved aspartates, Asp28 and Asp506, in the N- and C-terminal signature sequences, respectively, of human PiT2 to asparagine and analyzed P(i) uptake function in Xenopus laevis oocytes. Both mutant proteins were expressed at the cell surface of the oocytes but exhibited knocked out NaP(i) transport function. Human PiT2 is also a retroviral receptor and we have previously shown that this function can be exploited as a control for proper processing and folding of mutant proteins. Both mutant transporters displayed wild-type receptor functions implying that their overall architecture is undisturbed. Thus the presence of an aspartic acid in either of the PiT family signature sequences is critical for the Na+-dependent P(i) transport function of human PiT2. The conservation of the aspartates among proteins using either Na+- or H+-gradients for P(i) transport suggests that they are involved in H+-dependent P(i) transport as well. Current results favor a membrane topology model in which the N- and C-terminal PiT family signature sequences are positioned in intra- and extracellular loops, respectively, suggesting that they are involved in related functions on either side of the membrane. The present data are in agreement with a possible role of the signature sequences in translocation of cations.

High-resolution x-ray crystal structures of the villin headpiece subdomain, an ultrafast folding protein.

The 35-residue subdomain of the villin headpiece (HP35) is a small ultrafast folding protein that is being intensely studied by experiments, theory, and simulations. We have solved the x-ray structures of HP35 and its fastest folding mutant [K24 norleucine (nL)] to atomic resolution and compared their experimentally measured folding kinetics by using laser temperature jump. The structures, which are in different space groups, are almost identical to each other but differ significantly from previously solved NMR structures. Hence, the differences between the x-ray and NMR structures are probably not caused by lattice contacts or crystal/solution differences, but reflect the higher accuracy of the x-ray structures. The x-ray structures reveal important details of packing of the hydrophobic core and some additional features, such as cross-helical H bonds. Comparison of the x-ray structures indicates that the nL substitution produces only local perturbations. Consequently, the finding that the small stabilization by the mutation is completely reflected in an increased folding rate suggests that this region of the protein is as structured in the transition state as in the folded structure. It is therefore a target for engineering to increase the folding rate of the subdomain from approximately 0.5 micros(-1) for the nL mutant to the estimated theoretical speed limit of approximately 3 micros(-1).

Folding Efficiency Is Rate-limiting in Dopamine D4 Receptor Biogenesis

Dopamine receptors are G protein-coupled receptors that are critically involved in locomotion, reward, and cognitive processes. The D2 class of dopamine receptors (DRD2, -3, and -4) is the target for antipsychotic medication. DRD4 has been implicated in cognition, and genetic studies have found an association between a highly polymorphic repeat sequence in the human DRD4 coding region and attention deficit hyperactivity disorder. Using DRD4 as a model, we show that antipsychotics can function as potent pharmacological chaperones up-regulating receptor expression and can also rescue a non-functional DRD4 folding mutant. This chaperone-mediated up-regulation involves reduced degradation by the 26 S proteasome; likely via the stabilization of newly synthesized receptor in the endoplasmic reticulum. Dopamine itself can function as a chaperone when shuttled into the cell by means of the dopamine transporter. Furthermore, different repeat variants of DRD4 display differential sensitivity to this chaperone effect. These data suggest that folding efficiency may be rate-limiting for dopamine receptor biogenesis and that this efficiency differs between receptor variants. Consequently, the clinical profile of dopaminergic ligands, including antipsychotics, may include their ability to serve as pharmacological chaperones.

817. Protein Transduction Domain Mediated DFRD Correction of CFTR Function in Airway Cells from DF508 Homozygous CF Patients

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), associated with a loss of cAMP-regulated anion conductance at epithelial cell apical membranes. The common folding mutant, DF508CFTR, is retained in the endoplasmic reticulum (ER) and is degraded by ubiquitin-proteasome pathways. DF508CFTR retention in the ER involves prolonged association with molecular chaperones. We have shown that adenovirus mediated gene expression of the first nucleotide-binding domain plus the regulatory domain (RD) from DF508CFTR (DFRD) facilitates the biogenesis of mature, full-length DF508CFTR protein and elicits cAMP-stimulated anion transport in primary human bronchial epithelial cells isolated from homozygous DF508 CF patients (DF/DF cells). However, viral mediated gene delivery into airway tissue suffers from low infection efficiencies. Recently, we have identified a class of cationic protein transduction domains (PTDs), able to transduce variety of cells and tissues in vitro and in vivo, including human airway cells and mouse airway epithelial cells, and another non-cationic class of PTDs, such as AWP1, specifically targeting to airway tissue from M13 phage display library. Fusion of DFRD with the cationic PTD-5 evokes a dose-dependent, cAMP-stimulated anion efflux in DF/DF cells. The co-chaperone, Hdj2, selectively binds DF508CFTR. Expression of DFRD produced a dose-dependent decrease in the DF508CFTR-Hdj2 interaction and in |[Delta]|F508CFTR protein aggregation. The PTD-5-DFRD fusion protein blocked this interaction in a dose-dependent manner. These data indicate that disruption of the interactions of Hdj2 with DF508CFTR by gene and protein transduction-mediated delivery of DFRD restores its maturation, trafficking and regulated anion channel activity. These results suggest that protein transduction domain-mediated DFRD fragment delivery may provide a therapy for CF.