The short apical membrane half-life of rescued {Delta}F508-cystic fibrosis transmembrane conductance regulator (CFTR) results from accelerated endocytosis of {Delta}F508-CFTR in polarized human airway epithelial cells

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in individuals with cystic fibrosis, DeltaF508, causes retention of DeltaF508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl(-) channels in the apical plasma membrane. Rescue of DeltaF508-CFTR by reduced temperature or chemical means reveals that the DeltaF508 mutation reduces the half-life of DeltaF508-CFTR in the apical plasma membrane. Because DeltaF508-CFTR retains some Cl(-) channel activity, increased expression of DeltaF508-CFTR in the apical membrane could serve as a potential therapeutic approach for cystic fibrosis. However, little is known about the mechanisms responsible for the short apical membrane half-life of DeltaF508-CFTR in polarized human airway epithelial cells. Accordingly, the goal of this study was to determine the cellular defects in the trafficking of rescued DeltaF508-CFTR that lead to the decreased apical membrane half-life of DeltaF508-CFTR in polarized human airway epithelial cells. We report that in polarized human airway epithelial cells (CFBE41o-) the DeltaF508 mutation increased endocytosis of CFTR from the apical membrane without causing a global endocytic defect or affecting the endocytic recycling of CFTR in the Rab11a-specific apical recycling compartment.     

Role for phospholipid interactions in the trafficking defect of Delta F508-CFTR

Cystic fibrosis commonly occurs as a consequence of the DeltaF508 mutation in the first nucleotide binding fold domain (NBF-1) of CFTR. The mutation causes retention of the mutant CFTR molecule in the endoplasmic reticulum, and this aberrant trafficking event is believed to be due to defective interactions between the mutant NBF-1 domain and other cellular factors in the endoplasmic reticulum. Since the NBF-1 domain has been shown to interact with membranes, we wanted to investigate whether NBF-1 and CFTR interactions with specific phospholipid chaperones might play a role in trafficking. We have found that the recombinant wild-type NBF-1 interacts selectively with phosphatidylserine (PS) rather than phosphatidylcholine (PC). By contrast, NBF-1 carrying the DeltaF508 mutation loses the ability to discriminate between these two phospholipids. In cells expressing DeltaF508-CFTR, replacement of PC by noncharged analogues results in an absolute increase in CFTR expression. In addition, we detected progressive expression of higher molecular weight CFTR forms. Thus, phospholipid chaperones may be important for CFTR trafficking, and contribute to the pathology of cystic fibrosis.     

Nanomolar affinity small molecule correctors of defective Delta F508-CFTR chloride channel gating

Deletion of Phe-508 (Delta F508) is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) causing cystic fibrosis. Delta F508-CFTR has defects in both channel gating and endoplasmic reticulum-to-plasma membrane processing. We identified six novel classes of high affinity potentiators of defective Delta F508-CFTR Cl- channel gating by screening 100,000 diverse small molecules. Compounds were added 15 min prior to assay of iodide uptake in epithelial cells co-expressing Delta F508-CFTR and a high sensitivity halide indicator (YFP-H148Q/I152L) in which Delta F508-CFTR was targeted to the plasma membrane by culture at 27 degrees C for 24 h. Thirty-two compounds with submicromolar activating potency were identified; most had tetrahydrobenzothiophene, benzofuran, pyramidinetrione, dihydropyridine, and anthraquinone core structures (360-480 daltons). Further screening of >1000 structural analogs revealed tetrahydrobenzothiophenes that activated DeltaF508-CFTR Cl- conductance reversibly with Kd < 100 nm. Single-cell voltage clamp analysis showed characteristic CFTR currents after Delta F508-CFTR activation. Activation required low concentrations of a cAMP agonist, thus mimicking the normal physiological response. A Bayesian computational model was developed using tetrahydrobenzothiophene structure-activity data, yielding insight into the physical character and structural features of active and inactive potentiators and successfully predicting the activity of structural analogs. Efficient potentiation of defective Delta F508-CFTR gating was also demonstrated in human bronchial epithelial cells from a Delta F508 cystic fibrosis subject after 27 degrees C temperature rescue. In conjunction with correctors of defective Delta F508-CFTR processing, small molecule potentiators of defective Delta F508-CFTR gating may be useful for therapy of cystic fibrosis caused by the Delta F508 mutation.     

Trafficking of GFP-tagged Delta F508-CFTR to the plasma membrane in a polarized epithelial cell line

The F508 mutationreduces the amount of cystic fibrosis transmembrane conductanceregulator (CFTR) expressed in the plasma membrane of epithelial cells.However, a reduced temperature, butyrate compounds, and "chemicalchaperones" allow F508-CFTR to traffic to the plasma membrane andincrease Cl permeability in heterologous and nonpolarizedcells. Because trafficking is affected by the polarized state ofepithelial cells and is cell-type dependent, our goal was to determinewhether these maneuvers induce F508-CFTR trafficking to the apicalplasma membrane in polarized epithelial cells. To this end, wegenerated and characterized a line of polarized Madin-Darby caninekidney (MDCK) cells stably expressing F508-CFTR tagged with greenfluorescent protein (GFP). A reduced temperature, glycerol, butyrate,or DMSO had no effect on 8-(4-chlorophenylthio)-cAMP(CPT-cAMP)-stimulated transepithelial Cl secretion acrosspolarized monolayers. However, when the basolateral membrane waspermeabilized, butyrate, but not the other experimental maneuvers,increased the CPT-cAMP-stimulated Cl current across theapical plasma membrane. Thus butyrate increased the amount offunctional F508-CFTR in the apical plasma membrane. Butyrate failedto stimulate transepithelial Cl secretion because ofinhibitory effects on Cl uptake across the basolateralmembrane. These observations suggest that studies on heterologous andnonpolarized cells should be interpreted cautiously. The GFP tag onF508-CFTR will allow investigation of F508-CFTR trafficking inliving, polarized MDCK epithelial cells in real time.

     

Genistein restores functional interactions between Delta F508-CFTR and ENaC in Xenopus oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its Cl(-) channel properties, has regulatory interactions with other epithelial ion channels including the epithelial Na(+) channel (ENaC). Both the open probability and surface expression of wild type CFTR Cl(-) channels are increased significantly when CFTR is co-expressed in Xenopus oocytes with alphabetagamma-ENaC, and conversely, the activity of ENaC is inhibited following wild type CFTR activation. Using the Xenopus oocyte expression system, a lack of functional regulatory interactions between DeltaF508-CFTR and ENaC was observed following activation of DeltaF508-CFTR by forskolin and isobutylmethylxanthine (IBMX). Whole cell currents in oocytes expressing ENaC alone decreased in response to genistein but increased in response to a combination of forskolin and IBMX followed by genistein. In contrast, ENaC currents in oocytes co-expressing ENaC and DeltaF508-CFTR remained stable following stimulation with forskolin/IBMX/genistein. Furthermore, co-expression of DeltaF508-CFTR with ENaC enhanced the forskolin/IBMX/genistein-mediated activation of DeltaF508-CFTR. Our data suggest that genistein restores regulatory interactions between DeltaF508-CFTR and ENaC and that combinations of protein repair agents, such as 4-phenylbutyrate and genistein, may be necessary to restore DeltaF508-CFTR function in vivo.     

Cross-linking of F508-CFTR promotes its trafficking to the plasma membrane

CFTR is a cAMP-activated chloride channel responsible for agonist stimulated chloride and fluid transport across epithelial surfaces.¹ Mutations in the CFTR gene lead to cystic fibrosis (CF) which affects the function of secretory organs like the intestine, the pancreas, the airways and the sweat glands. Most of the morbidity and mortality in CF has been linked to a decrease in airway function.² The ΔF508 mutation is the most common CF-related mutation in the Caucasian population and represents 90% of CF alleles. Homozygote carriers of this mutation present with a severe CF phenotype.³ The ΔF508 mutation causes misfolding of the nascent CFTR polypeptide, which leads to inefficient export from the endoplasmic reticulum (ER) and rapid degradation by the proteasome.⁴Key words: cystic fibrosis, endoplasmic reticulum, oligomer, processing mutation, curcuminGiven the frequency of the ΔF508 processing mutation and the severity of its corresponding phenotype, much research has focused on identifying compounds that restore the trafficking and function of this mutant at the plasma membrane. Several synthetic ‘correctors’ of ΔF508 mis-processing and ‘potentiators’ of mutant channel activity have been identified.^5,6 Natural compounds such as curcumin also have generated interest. Curcumin is an organic phenolic compound abundant in turmeric, an Indian spice extracted from the rhizome of Curcuma longa.⁷ Earlier studies performed using ΔF508/ΔF508 mouse models and human airway epithelial cell lines suggested that curcumin may act as a ΔF508-CFTR trafficking corrector.⁸ Also, we and others showed that curcumin stimulates CFTR channel activity in excised membrane patches.^9,10 This stimulation occurs in the absence of ATP binding, which is normally required for channel opening.¹⁰ Binding sites of correctors and potentiators within the CFTR polypeptide as well as the molecular mechanisms underlying the rescue of CFTR trafficking and function remain to be elucidated. In our attempt to understand how curcumin could circumvent the normally critical step of ATP binding to promote CFTR channel activity we investigated the effect of curcumin on CFTR conformation by using biochemical assays. We showed that curcumin caused dimerization of several CFTR channel constructs (including ΔF508-CFTR) in a dose- and time-dependent manner both in microsomes and within intact cells. This effect of curcumin on CFTR oligomerization is attributable to its reactive β-diketone groups, which may undergo an oxidation reaction with CFTR nucleophilic amino acid residues.¹¹ Importantly, CFTR channel activation by curcumin is unrelated to its cross-linking effect. We identified cyclic derivatives of curcumin that lack this cross-linking activity but still promote CFTR channel function.¹¹Here we examined the possibility that the cross-linking of ΔF508-CFTR channels by curcumin promotes the delivery of this ER processing mutant to the cell surface. We were motivated to test this possibility for three reasons: (i) our previous evidence that curcumin-induced dimers of wild-type CFTR polypeptides were detected at the cell surface where they remained over an hour after the removal of curcumin;¹¹ (ii) the very efficient cross-linking of the immature (ER) forms of wild-type CFTR and the ΔF508-CFTR mutant that we observed earlier¹¹ and (iii) prior evidence from our group that the ER export and cell surface delivery of ΔF508-CFTR polypeptides could be promoted by the co-expression of this mutant with certain CFTR fragments (trans-complementation).¹² The latter result might be due to the existence of ER retention ‘signals’ that are exposed on the ΔF508-CFTR polypeptide but become buried by interacting (complementing) fragments.Figure 1 provides evidence that ΔF508-CFTR oligomers that form in response to curcumin treatment do indeed appear at the surfaces of cultured airway epithelial cells (CF bronchial epithelial (CFBE) cells stably transfected with this CFTR mutant). Surface biotinylation assays were performed to detect the appearance of ΔF508-CFTR polypeptides at the cell surface. MESNA, a cell impermeant reducing agent that cleaves the biotin label, was used to verify the surface accessibility of the labeled ΔF508-CFTR polypeptides. ΔF508-CFTR polypeptides were precipititated with streptavidinagarose (surface pool) or with a CFTR monoclonal antibody (total pool). In the absence of curcumin treatment the great majority of the ΔF508-CFTR protein existed as the ER form (monomeric band B), as previously observed by many investigators (Fig. 1, lane 5). No band B was detected in the surface pool before or after curcumin treatment (Fig. 1, lanes 1, 2). As we reported earlier, treatment of the cells with 50 µM curcumin for 15 mins at 37°C cross-linked nearly all of the ΔF508-CFTR polypeptides into higher order complexes (e.g., dimers, termed band D here; lanes 6–8 in Fig. 1). Interestingly, these higher order forms of ΔF508-CFTR were readily apparent in the surface pool (Fig. 1, lane 2).Open in a separate windowFigure 1ΔF508-CFTR oligomers detected at the surfaces of airway epithelial cells after curcumin treatment. ΔF508-CFTR expressing CFBE cells were treated with curcumin (50 µM) for 15 min at 37°C. Cell surface proteins were then biotinylated (Sulfo-NHS-SS-Biotin, 1 mg/ml) for 30 min at 4°C followed by cell lysis with 1% Triton X-100. Surface proteins were isolated by streptavidin pulldown and ΔF508-CFTR was isolated from the total cell protein pool by immunoprecipitation with an anti-CFTR C-terminus antibody (clone 24-1, R&D systems). After SDS-PAGE the ΔF508-CFTR signal was detected by immunoblotting using the 24-1 antibody described above. (SP: streptavidin pulldown; IP: immunoprecipitation). As an additional control curcumin-treated cells were treated with the cell impermeant MESNA after biotinylation to strip the biotin off the cell surface proteins with which it had reacted.CFTR oligomers also can be generated by standard chemical cross-linkers such as DSS, as previously reported by others and confirmed by us.¹³ Figure 2 shows that oligomers of ΔF508-CFTR that are induced by DSS treatment also appear in the surface pool. These experiments were performed using transiently transfected HEK-293T cells with 30 µM curcumin as a positive control. Quantitative densitometry results are shown in Figure 3. By titrating the DSS concentration we observed a dose-dependent disappearance of the monomeric band B form, a corresponding increase in the band D (dimer) pool and the appearance of higher order oligomers (band E) which prevailed at higher DSS concentrations (see total cell pool data in right-hand). A small amount of the band D form was detected in the absence of DSS or curcumin treatment, which might represent some spontaneous cross-linking of ΔF508-CFTR polypeptides under these conditions. The DSS and curcumin-induced ΔF508-CFTR oligomers were readily detected in the surface pool. The densitometry analysis revealed that 20 ± 5% and 33 ± 19% of the total oligomer pool (combined bands D and E) was found in the surface pool after treatment with 0.1 mM DSS (n = 3) or 30 µM curcumin (n = 3), respectively, which corresponded to a 17 ± 7 and 26 ± 20 fold increase compared to the control condition (i.e., no DSS or no curcumin).Open in a separate windowFigure 2ΔF508-CFTR oligomers detected at the surfaces of HEK cells after DSS or curcumin treatment. ΔF508-CFTR expressing HEK cells were treated with the indicated concentrations of DSS or with 30 µM curcumin (*) for 15 min at 37°C. Cell surface proteins were then biotinylated and isolated by streptavidin pulldown as described above. ΔF508-CFTR was immunoprecipitated from the total cell protein pool with the 24-1 antibody and detected by immunoblotting as before (SP: streptavidin pulldown; IP: immunoprecipitation). Band B corresponds to ΔF508 monomer (ER form). Band D corresponds to ΔF508 dimer. Band E corresponds to a higher degree of ΔF508 oligomerization. Each panel corresponds to a different exposure of the same blot.Open in a separate windowFigure 3Dose-dependent expression of ΔF508-CFTR oligomers at the surfaces of HEK cells after DSS treatment. CFTR signals detected by the 24-1 antibody from three different experiments as the one described in Figure 2 were analyzed using the ImageJ software (from the National Institute of Health). (A) band B signal intensity is plotted as a function of the DSS concentrations. Signals analyzed correspond to ΔF508-CFTR band B immunoprecipitated by the 24-1 antibody. (B) band D plus band E signal intensities are plotted as a function of the DSS concentration. Signals analyzed correspond to the sum of ΔF508-CFTR band D and band E immunoprecipitated by the 24-1 antibody. (C) band D plus band E signal intensities at the cell surface are plotted as a function of the DSS concentration. Signals analyzed correspond to the sum of ΔF508-CFTR band D and band E isolated from the surfaces of ΔF508-CFTR expressing HEK cells by biotinylation and streptavidin pulldown. (D) the ratio between the amount of band E and D at the surfaces of ΔF508-CFTR expressing HEK cells is plotted as a function of the DSS concentration. Error bars are SEMs.Altogether these data indicate that the cross-linking of ΔF508-CFTR band B into oligomers by curcumin or DSS allows ΔF508-CFTR to traffic to the cell surface. This effect might be caused by the burial of ER retention motifs within the oligomer, which also could explain our previous trans-complementation results in which we observed that certain CFTR fragments promote the cell surface delivery of this processing mutant.¹² Although non-specific protein cross-linkers like DSS would not be therapeutically beneficial, more specific CFTR cross-linkers (perhaps curcumin?) may be worth considering for treating CF disease linked to ER processing mutations in CFTR. In this regard, we note that cross-linked CFTR polypeptides appear to retain chloride channel activity. Namely, in our prior excised patch clamp studies we observed stable CFTR channel activity when these patches were exposed to curcumin at doses and times that promote robust cross-linking of CFTR polypeptides.^10,11     

Calcium-pump inhibitors induce functional surface expression of Delta F508-CFTR protein in cystic fibrosis epithelial cells

The most common mutation in cystic fibrosis, Delta F508, results in a cystic fibrosis transmembrane conductance regulator (CFTR) protein that is retained in the endoplasmic reticulum (ER). Retention is dependent upon chaperone proteins, many of which require Ca(++) for optimal activity. Interfering with chaperone activity by depleting ER Ca(++) stores might allow functional Delta F508-CFTR to reach the cell surface. We exposed several cystic fibrosis cell lines to the ER Ca(++) pump inhibitor thapsigargin and evaluated surface expression of Delta F508-CFTR. Treatment released ER-retained Delta F508-CFTR to the plasma membrane, where it functioned effectively as a Cl(-) channel. Treatment with aerosolized calcium-pump inhibitors reversed the nasal epithelial potential defect observed in a mouse model of Delta F508-CFTR expression. Thus, ER calcium-pump inhibitors represent a potential target for correcting the cystic fibrosis defect.     

A foldable CFTR{Delta}F508 biogenic intermediate accumulates upon inhibition of the Hsc70-CHIP E3 ubiquitin ligase

CFTRDeltaF508 exhibits a correctable protein-folding defect that leads to its misfolding and premature degradation, which is the cause of cystic fibrosis (CF). Herein we report on the characterization of the CFTRDeltaF508 biogenic intermediate that is selected for proteasomal degradation and identification of cellular components that polyubiquitinate CFTRDeltaF508. Nonubiquitinated CFTRDeltaF508 accumulates in a kinetically trapped, but folding competent conformation, that is maintained in a soluble state by cytosolic Hsc70. Ubiquitination of Hsc70-bound CFTRDeltaF508 requires CHIP, a U box containing cytosolic cochaperone. CHIP is demonstrated to function as a scaffold that nucleates the formation of a multisubunit E3 ubiquitin ligase whose reconstituted activity toward CFTR is dependent upon Hdj2, Hsc70, and the E2 UbcH5a. Inactivation of the Hsc70-CHIP E3 leads CFTRDeltaF508 to accumulate in a nonaggregated state, which upon lowering of cell growth temperatures, can fold and reach the cell surface. Inhibition of CFTRDeltaF508 ubiquitination can increase its cell surface expression and may provide an approach to treat CF.     

4'-Methyl-4,5'-bithiazole-based correctors of defective delta F508-CFTR cellular processing

The synthesis and Delta F508-CFTR corrector activity of a 148-member methylbithiazole-based library are reported. Synthetic routes were devised and optimized to generate methylbithiazole analogs in four steps. Corrector potency and efficacy were assayed using epithelial cells expressing human Delta F508-CFTR. These structure-activity data establish that the bithiazole substructure plays a critical function; eight novel methylbithiazole correctors were identified with low micromolar potencies.     

Activation of Delta F508 CFTR in an epithelial monolayer

The F508 mutation leads to retention of cystic fibrosistransmembrane conductance regulator (CFTR) in the endoplasmic reticulum and rapid degradation by the proteasome and other proteolytic systems.In stably transfected LLC-PK₁(porcine kidney) epithelial cells, F508 CFTR conforms to thisparadigm and is not present at the plasma membrane. WhenLLC-PK₁ cells or human nasal polyp cells derived from a F508 homozygous patient are grown on plastic dishes and treated with an epithelial differentiating agent (DMSO, 2%for 4 days) or when LLC-PK₁ cellsare grown as polarized monolayers on permeable supports, plasmamembrane F508 CFTR is significantly increased. Moreover, whenconfluent LLC-PK₁ cells expressingF508 CFTR were treated with DMSO and mounted in an Ussing chamber, afurther increase in cAMP-activated short-circuit current (i.e., ~7µA/cm²;P < 0.00025 compared with untreatedcontrols) was observed. No plasma membrane CFTR was detected after DMSOtreatment in nonepithelial cells (mouse L cells) expressing F508CFTR. The experiments describe a way to augment F508 CFTR maturationin epithelial cells that appears to act through a novel mechanism andallows insertion of functional F508 CFTR in the plasma membranes oftransporting cell monolayers. The results raise the possibility thatincreased epithelial differentiation might increase the delivery ofF508 CFTR from the endoplasmic reticulum to the Golgi, where theF508 protein is shielded from degradative pathways such as theproteasome and allowed to mature.

     

Crystallin {gamma}B-I4F mutant protein binds to {alpha}-crystallin and affects lens transparency

A new mouse mutant line, Clapper, identified from N-ethyl-N-nitrosurea (ENU)-mutagenized mice, develops a dominant lamellar cataract. The cataract blocks the image of retinal fundus and transmits a fuzzy fluorescein image of retinal vasculature during angiography. The cataractous lens opacity decreases as the mice age. The Clapper mutation has been identified to be a missense mutation of the gammaB-crystallin gene that replaces the 4th isoleucine residue with a phenylalanine (gammaB-I4F). Unlike wild type gammaB, the gammaB-I4F mutant protein binds to alpha-crystallin to form high molecular weight complexes in vivo and in vitro. Circular dichroism measurements indicate that gammaB-I4F protein is less stable than wild type gammaB at high temperature. Darkly stained aggregates, enlarged interfiber spaces, and disorganized and smaller inner mature fibers were found in the regions of the cataract in homozygous Clapper mutant lenses. Thus, the lamellar cataract is likely due to the light-scattering effects of the enlarged interfiber spaces and protein aggregates caused by gammaB-I4F mutant proteins interacting with alpha-crystallin in the lens.     

Altered biogenesis of deltaF508-CFTR following treatment with doxorubicin.

Cystic fibrosis (CF) is caused by mutations to the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common of these mutations is deletion of a phenylalanine residue at position 508 (Delta F508), which accounts for approximately 70% of all CF alleles. This mutation interferes with the biogenesis and maturation of Delta F508-CFTR to the plasma membrane. However, Delta F508-CFTR can partially function upon proper localization. Thus, pharmacological correction of Delta F508-CFTR maturation holds promise in CF therapy. Our previous studies indicate that a single non-cytotoxic dose of the anthracycline doxorubicin (Dox) significantly increase Delta F508-CFTR-associated chloride secretion in MDCK cells by increasing the expression of this protein at the apical plasma membrane. We report here that Dox alters the biogenesis of Delta F508-CFTR. Treatment with Dox increases the resistance of Delta F508-CFTR to trypsin digestion, possibly by expediting protein folding. Further, treatment with Dox reduces the amount of polyubiquitinated Delta F508-CFTR in cells and prolongs the half-life of this protein. Concomitantly, treatment with Dox decreases the association of Delta F508-CFTR with HSP70 but does not alter the expression of major HSP70 family members. Based on these results, we propose that Dox expedites the folding and maturation of Delta F508-CFTR by acting as a pharmacological chaperone, which consequently promotes the functional expression of this protein in MDCK cells.     

The dominant negative thyroid hormone receptor beta-mutant {Delta}337T alters PPAR{alpha} signaling in heart

PPARalpha and TR independently regulate cardiac metabolism. Although ligands for both these receptors are currently under evaluation for treatment of congestive heart failure, their interactions or signaling cooperation have not been investigated in heart. We tested the hypothesis that cardiac TRs interact with PPARalpha regulation of target genes and used mice exhibiting a cardioselective Delta337T TRbeta1 mutation (MUT) to reveal cross-talk between these nuclear receptors. This dominant negative transgene potently inhibits DNA binding for both wild-type (WT) TRalpha and TRbeta. We used UCP3 and MTE-1 as principal reporters and analyzed gene expression from hearts of transgenic (MUT) and nontransgenic (WT) littermates 6 h after receiving either specific PPARalpha ligand (WY-14643) or vehicle. Interactions were determined through qRT-PCR analyses, and the extent of these interactions across multiple genes was determined using expression arrays. In the basal state, we detected no differences between groups for protein content for UCP3, PPARalpha, TRalpha2, RXRbeta, or PGC-1alpha. However, protein content for TRalpha1 and the PPARalpha heterodimeric partner RXRalpha was diminished in MUT, whereas PPARbeta increased. We demonstrated cross-talk between PPAR and TR for multiple genes, including the reporters UCP3 and MTE1. WY-14643 induced a twofold increase in UCP3 gene expression that was totally abrogated in MUT. We demonstrated variable cross-talk patterns, indicating that multiple mechanisms operate according to individual target genes. The non-ligand-binding TRbeta1 mutation alters expression for multiple nuclear receptors, providing a novel mechanism for interaction that has not been previously demonstrated. These results indicate that therapeutic response to PPARalpha ligands may be determined by thyroid hormone state and TR function.     

A small protein required for the switch from {sigma}F to {sigma}G during sporulation in Bacillus subtilis

A cascade of alternative sigma factors governs the program of developmental gene expression during sporulation in Bacillus subtilis. Little is known, however, about how the early-acting sigma factors are inactivated and replaced by the later-acting factors. Here we identify a small protein, Fin (formerly known as YabK), that is required for efficient switching from σ(F)- to σ(G)-directed gene expression in the forespore compartment of the developing sporangium. The fin gene, which is conserved among Bacillus species and species of related genera, is transcribed in the forespore under the control of both σ(F) and σ(G). Cells mutant for fin are unable to fully deactivate σ(F) and, conversely, are unable to fully activate σ(G). Consistent with their deficiency in σ(G)-directed gene expression, fin cells are arrested in large numbers following the engulfment stage of sporulation, ultimately forming 50-fold fewer heat-resistant spores than the wild type. Based in part on the similarity of Fin to the anti-σ(G) factor CsfB (also called Gin), we speculate that Fin is an anti-σ(F) factor which, by disabling σ(F), promotes the switch to late developmental gene expression in the forespore.     

Mutational Analysis of the Pentose Phosphate and Entner-Doudoroff Pathways in Gluconobacter oxydans Reveals Improved Growth of a {Delta}edd {Delta}eda Mutant on Mannitol

The obligatory aerobic acetic acid bacterium Gluconobacter oxydans 621H oxidizes sugars and sugar alcohols primarily in the periplasm, and only a small fraction is metabolized in the cytoplasm. The latter can occur either via the Entner-Doudoroff pathway (EDP) or via the pentose phosphate pathway (PPP). The Embden-Meyerhof pathway is nonfunctional, and a cyclic operation of the tricarboxylic acid cycle is prevented by the absence of succinate dehydrogenase. In this work, the cytoplasmic catabolism of fructose formed by oxidation of mannitol was analyzed with a Δgnd mutant lacking the oxidative PPP and a Δedd Δeda mutant devoid of the EDP. The growth characteristics of the two mutants under controlled conditions with mannitol as the carbon source and enzyme activities showed that the PPP is the main route for cytoplasmic fructose catabolism, whereas the EDP is dispensable and even unfavorable. The Δedd Δeda mutant (lacking 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase) formed 24% more cell mass than the reference strain. In contrast, deletion of gnd (6-phosphogluconate dehydrogenase) severely inhibited growth and caused a strong selection pressure for secondary mutations inactivating glucose-6-phosphate dehydrogenase, thus preventing fructose catabolism via the EDP also. These Δgnd zwf* mutants (with a mutation in the zwf gene causing inactivation of the glucose-6-phosphate dehydrogenase) were almost totally disabled in fructose catabolism but still produced about 14% of the carbon dioxide of the reference strain, possibly by catabolizing substrates from the yeast extract. Overexpression of gnd in the reference strain improved biomass formation in a similar manner as deletion of edd and eda, further confirming the importance of the PPP for cytoplasmic fructose catabolism.     

Mild processing defect of porcine DeltaF508-CFTR suggests that DeltaF508 pigs may not develop cystic fibrosis disease

Recent efforts have made significant progress in generating transgenic pigs with the ΔF508-CFTR mutation to model the lung and pancreatic disease of human cystic fibrosis. However, species differences in the processing and function of human, pig and mouse ΔF508-CFTR reported recently raise concerns about the phenotypic consequence of the gene-targeted pig model. The purpose of the present study was to characterize the ΔF508 mutant of porcine CFTR to evaluate the severity of its processing defect. Biochemical and immunofluorescence analysis in transfected COS7 and FRT cells indicated that pig ΔF508-CFTR efficiently targets to the plasma membrane and is present mainly as the mature glycosylated protein. Functional characterization in stably transfected FRT cells by fluorometric and electrophysiological assays supported efficient plasma membrane targeting of pig ΔF508-CFTR. The mild cellular processing defect of pig ΔF508-CFTR suggests that its gene-targeted pig model may not develop the lung and pancreatic phenotypes seen in CF patients.