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Background: Most cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that lead to protein misfolding and degradation by the ubiquitin-proteasome system. Previous studies demonstrated that PIAS4 facilitates the modification of wild-type (WT) and F508del CFTR by small ubiquitin-like modifier (SUMO)-1, enhancing CFTR biogenesis by slowing immature CFTR degradation and producing increased immature CFTR band B. Methods: We evaluated two correction strategies using misfolding mutants, including the common variant, F508del. We examined the effects on mutant expression of co-expression with PIAS4 (E3 SUMO ligase), and/or the corrector, C18. To study the impact of these correction conditions, we transfected CFBE410- cells, a bronchial epithelial cell line, with a CFTR mutant plus: (1) empty vector, (2) empty vector plus overnight 5 µM C18, (3) PIAS4, and (4) PIAS4 plus C18. We assessed expression at steady state by immunoblot of CFTR band B, and if present, band C, and the corresponding C:B band ratio. The large PIAS4-induced increase in band B expression allowed us to ask whether C18 could act on the now abundant immature protein to enhance correction above the control level, as reported by the C:B ratio. Results: The data fell into three mutant CFTR categories as follows: (1) intransigent: no observable band C under any condition (i.e., C:B = 0); (2) throughput responsive: a C:B ratio less than control, but suggesting that the increased band C resulted from PIAS4-induced increases in band B production; and (3) folding responsive: a C:B ratio greater than control, reflecting C18-induced folding greater than that expected from increased throughput due to the PIAS4-induced band B level. Conclusion: These results suggest that the immature forms of CFTR folding intermediates occupy different loci within the energetic/kinetic folding landscape of CFTR. The evaluation of their properties could assist in the development of correctors that can target the more difficult-to-fold mutant conformations that occupy different sites within the CFTR folding pathway.
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Aberrant anion secretion across the bronchial epithelium is associated with airway disease, most notably in cystic fibrosis. Although the cystic fibrosis transmembrane conductance regulator (CFTR) is recognized as the primary source of airway anion secretion, alternative anion transport mechanisms play a contributing role. An alternative anion transporter of growing interest is SLC26A9, a constitutively active chloride channel that has been shown to interact with CFTR and may also contribute to bicarbonate secretion. Interest in SLC26A9 has been fueled by genome-wide association studies that suggest it is a significant modifier of CF disease severity. Despite this growing evidence that SLC26A9 plays an important role in the airway, its presence and function in bronchial epithelia remain poorly understood, in part, because its activity is difficult to separate from the activity of CFTR. Here, we present results using primary human bronchial epithelia (HBE) from multiple patient sources to confirm that SLC26A9 mRNA is present in HBE and that its constitutive channel activity is unaffected by knockdown of CFTR. Furthermore, SLC26A9 and CFTR show differential responses to common inhibitors of anion secretion. Finally, we assess the impact of bicarbonate on the activity of SLC26A9 and CFTR. These results confirm that SLC26A9 is the primary source of constitutive anion secretion across HBE, and should inform future studies focused on activation of SLC26A9 as an alternative anion channel in CF. These results should provide a strong foundation to investigate how single-nucleotide polymorphisms in SLC26A9 modulate airway disease.
Assuntos
Antiporters/metabolismo , Bicarbonatos/metabolismo , Brônquios/metabolismo , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Transportadores de Sulfato/metabolismo , Antiporters/genética , Antiporters/farmacologia , Transporte Biológico , Brônquios/efeitos dos fármacos , Células Cultivadas , Fibrose Cística/tratamento farmacológico , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/efeitos dos fármacos , Humanos , Transportadores de Sulfato/genéticaRESUMO
Cystic fibrosis (CF) occurs as a result of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which lead to misfolding, trafficking defects, and impaired function of the CFTR protein. Splicing factor proline/glutamine-rich (SFPQ) is a multifunctional nuclear RNA-binding protein (RBP) implicated in the regulation of gene expression pathways and intracellular trafficking. Here, we investigated the role of SFPQ in the regulation of the expression and function of F508del-CFTR in CF lung epithelial cells. We find that the expression of SFPQ is reduced in F508del-CFTR CF epithelial cells compared to WT-CFTR control cells. Interestingly, the overexpression of SFPQ in CF cells increases the expression as well as rescues the function of F508del-CFTR. Further, comprehensive transcriptome analyses indicate that SFPQ plays a key role in activating the mutant F508del-CFTR by modulating several cellular signaling pathways. This is the first report on the role of SFPQ in the regulation of expression and function of F508del-CFTR in CF lung disease. Our findings provide new insights into SFPQ-mediated molecular mechanisms and point to possible novel epigenetic therapeutic targets for CF and related pulmonary diseases.
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Brônquios/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Fator de Processamento Associado a PTB/fisiologia , Brônquios/patologia , Células Cultivadas , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica/fisiologia , Humanos , Mutação , TranscriptomaRESUMO
A growing number of diseases are linked to the misfolding of integral membrane proteins, and many of these proteins are targeted for ubiquitin-proteasome-dependent degradation. One such substrate is a mutant form of the Cystic Fibrosis Transmembrane Conductance Regulator (F508del-CFTR). Protein folding "correctors" that repair the F508del-CFTR folding defect have entered the clinic, but they are unlikely to protect the entire protein from degradation. To increase the pool of F508del-CFTR protein that is available for correction by existing treatments, we determined a structure-activity relationship to improve the efficacy and reduce the toxicity of an inhibitor of the E1 ubiquitin activating enzyme that facilitates F508del-CFTR maturation. A resulting lead compound lacked measurable toxicity and improved the ability of an FDA-approved corrector to augment F508del-CFTR folding, transport the protein to the plasma membrane, and maintain its activity. These data support a proof-of-concept that modest inhibition of substrate ubiquitination improves the activity of small molecule correctors to treat CF and potentially other protein conformational disorders.
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Benzoatos/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Furanos/farmacologia , Pirazóis/farmacologia , Ubiquitina/antagonistas & inibidores , Benzoatos/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Relação Dose-Resposta a Droga , Furanos/química , Humanos , Estrutura Molecular , Dobramento de Proteína/efeitos dos fármacos , Pirazóis/química , Relação Estrutura-Atividade , Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
ABC transporters transport a wealth of molecules across membranes and consist of transmembrane and cytosolic domains. Their activity cycle involves a tightly regulated and concerted domain choreography. Regulation is driven by the cytosolic domains and function by the transmembrane domains. Folding of these polytopic multidomain proteins to their functional state is a challenge for cells, which is mitigated by co-translational and sequential events. We here reveal the first stages of co-translational domain folding and assembly of CFTR, the ABC transporter defective in the most abundant rare inherited disease cystic fibrosis. We have combined biosynthetic radiolabeling with protease-susceptibility assays and domain-specific antibodies. The most N-terminal domain, TMD1 (transmembrane domain 1), folds both its hydrophobic and soluble helices during translation: the transmembrane helices pack tightly and the cytosolic N- and C-termini assemble with the first cytosolic helical loop ICL1, leaving only ICL2 exposed. This N-C-ICL1 assembly is strengthened by two independent events: (i) assembly of ICL1 with the N-terminal subdomain of the next domain, cytosolic NBD1 (nucleotide-binding domain 1); and (ii) in the presence of corrector drug VX-809, which rescues cell-surface expression of a range of disease-causing CFTR mutants. Both lead to increased shielding of the CFTR N-terminus, and their additivity implies different modes of action. Early assembly of NBD1 and TMD1 is essential for CFTR folding and positions both domains for the required assembly with TMD2. Altogether, we have gained insights into this first, nucleating, VX-809-enhanced domain-assembly event during and immediately after CFTR translation, involving structures conserved in type-I ABC exporters.
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Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Citosol/metabolismo , Biossíntese de Proteínas , Dobramento de Proteína , Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/biossíntese , Evolução Molecular , Genes Supressores , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Peptídeo Hidrolases/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Domínios Proteicos , Dobramento de Proteína/efeitos dos fármacos , Estrutura Secundária de ProteínaRESUMO
Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short lifespans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of approximately 40 days and died of gastrointestinal disease, but therapeutic regimens directed toward restoring gastrointestinal transit extended median survival to approximately 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression patterns in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF-like abnormalities in the bioelectric properties of the nasal and tracheal epithelia. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1-year-old CF rabbits as compared with WT littermates, and these were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.
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Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Animais , Sistemas CRISPR-Cas , Fibrose Cística/patologia , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Modelos Animais de Doenças , Feminino , Trato Gastrointestinal/patologia , Trato Gastrointestinal/fisiopatologia , Técnicas de Inativação de Genes , Humanos , Masculino , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Sistema Respiratório/patologia , Sistema Respiratório/fisiopatologia , Distribuição Tecidual , TranscriptomaRESUMO
BACKGROUND: Chronic inflammation is a hallmark among patients with cystic fibrosis (CF). We explored whether mutation-induced (F508del) misfolding of the cystic fibrosis transmembrane conductance regulator (CFTR), and/or secondary colonization with opportunistic pathogens, activate tissue remodeling and innate immune response drivers. METHODS: Using RNA-seq to interrogate global gene expression profiles, we analyzed stress response signaling cascades in primary human bronchial epithelia (HBE) and intestinal organoids. RESULTS: Primary HBE acquired from CF patients with advanced disease and prolonged exposure to pathogenic microorganisms display a clear molecular signature of activated tissue remodeling pathways, unfolded protein response (UPR), and chronic inflammation. Furthermore, CFTR misfolding induces inflammatory signaling cascades in F508del patient-derived organoids from both the distal small intestine and colon. CONCLUSION: Despite the small patient cohort size, this proof-of-principle study supports the use of RNA-seq as a means to both identify CF-specific signaling profiles in various tissues and evaluate disease heterogeneity. Our global transcriptomic data is a useful resource for the CF research community for analyzing other gene expression sets influencing CF disease signature but also transcriptionally contributing to CF heterogeneity.
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Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Fibrose Cística/microbiologia , Estresse do Retículo Endoplasmático/genética , Perfilação da Expressão Gênica , Imunidade Inata , Adulto , Brônquios/citologia , Células Cultivadas , Células Epiteliais , Feminino , Humanos , Inflamação , Pessoa de Meia-Idade , Organoides , Estudo de Prova de Conceito , Transdução de Sinais , Exacerbação dos Sintomas , TranscriptomaRESUMO
BACKGROUND: Recent advances in the functional analyses of endogenous non-coding RNA (ncRNA) molecules, including long non-coding RNAs (LncRNAs), have provided a new perspective on the crucial roles of RNA in gene regulation. Consequently, LncRNA deregulation is a key factor in various diseases, including pulmonary disorders like Cystic Fibrosis (CF). CF is the most common life limiting recessive disease in the U.S., and is due to mutations in the CFTR gene. CF mutations, of which the most common is F508del-CFTR, prevents correct folding, trafficking and function of the mutant CFTR protein and is further manifested by the hyper-expression of pro-inflammatory cytokines and chemokines into the airway lumen leading to bronchiectasis and culminating in lung destruction. METHODS: Here we report a distinct LncRNA signature and corresponding mRNAs that distinguishes CF lung (airway and parenchyma) tissues from matched non-CF controls (n = 4 each group), generated by microarray specific for LncRNAs which includes corresponding mRNA expressions. In silico analyses of the cellular processes that are impacted by these LncRNAs was performed using Gene Ontology (GO). A selected subset of LncRNAs were validated by quantitative real-time PCR. RESULTS: We have identified 636 LncRNAs differentially expressed in CF airway epithelium and 1974 in CF lung parenchyma compared to matched non-CF controls (fold change ≥2, p < 0.05), majority of which (> 50%) are intergenic. Interestingly, 15 of these differentially expressed LncRNAs and 9 coding mRNAs are common to airway and parenchyma tissues. GO analyses indicates that signaling pathways and cell membrane functions are significantly affected by the alteration in LncRNA expressions in CF lung tissues. Seven of the differentially expressed LncRNAs, exhibit similar expression trends in CFBE41o- compared to control cells. CONCLUSION: Understanding the mechanisms by which these LncRNAs regulate CF disease phenotype will help develop novel therapeutic targets for CF and related pulmonary diseases, such as COPD and Asthma.
Assuntos
Fibrose Cística/genética , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Pulmão/metabolismo , Tecido Parenquimatoso/metabolismo , RNA Longo não Codificante/genética , Transcriptoma , Adolescente , Adulto , Estudos de Casos e Controles , Linhagem Celular , Fibrose Cística/diagnóstico , Fibrose Cística/metabolismo , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Masculino , Adulto JovemRESUMO
Protein interactions that stabilize the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) at the apical membranes of epithelial cells have not yet been fully elucidated. We identified keratin 19 (CK19 or K19) as a novel CFTR-interacting protein. CK19 overexpression stabilized both wild-type (WT)-CFTR and Lumacaftor (VX-809)-rescued F508del-CFTR (where F508del is the deletion of the phenylalanine residue at position 508) at the plasma membrane (PM), promoting Cl- secretion across human bronchial epithelial (HBE) cells. CK19 prevention of Rab7A-mediated lysosomal degradation was a key mechanism in apical CFTR stabilization. Unexpectedly, CK19 expression was decreased by â¼40% in primary HBE cells from homogenous F508del patients with CF relative to non-CF controls. CK19 also positively regulated multidrug resistance-associated protein 4 expression at the PM, suggesting that this keratin may regulate the apical expression of other ATP-binding cassette proteins as well as CFTR.-Hou, X., Wu, Q., Rajagopalan, C., Zhang, C., Bouhamdan, M., Wei, H., Chen, X., Zaman, K., Li, C., Sun, X., Chen, S., Frizzell, R. A., Sun, F. CK19 stabilizes CFTR at the cell surface by limiting its endocytic pathway degradation.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Endocitose , Queratina-19/metabolismo , Proteólise , Fibrose Cística/genética , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células HEK293 , Células HeLa , Humanos , Queratina-19/genética , Lisossomos/genética , Lisossomos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mutação , Estabilidade ProteicaRESUMO
A pathway for cystic fibrosis transmembrane conductance regulator (CFTR) degradation is initiated by Hsp27, which cooperates with Ubc9 and binds to the common F508del mutant to modify it with SUMO-2/3. These SUMO paralogues form polychains, which are recognized by the ubiquitin ligase, RNF4, for proteosomal degradation. Here, protein array analysis identified the SUMO E3, protein inhibitor of activated STAT 4 (PIAS4), which increased wild-type (WT) and F508del CFTR biogenesis in CFBE airway cells. PIAS4 increased immature CFTR threefold and doubled expression of mature CFTR, detected by biochemical and functional assays. In cycloheximide chase assays, PIAS4 slowed immature F508del degradation threefold and stabilized mature WT CFTR at the plasma membrance. PIAS4 knockdown reduced WT and F508del CFTR expression by 40-50%, suggesting a physiological role in CFTR biogenesis. PIAS4 modified F508del CFTR with SUMO-1 in vivo and reduced its conjugation to SUMO-2/3. These SUMO paralogue-specific effects of PIAS4 were reproduced in vitro using purified F508del nucleotide-binding domain 1 and SUMOylation reaction components. PIAS4 reduced endogenous ubiquitin conjugation to F508del CFTR by â¼50% and blocked the impact of RNF4 on mutant CFTR disposal. These findings indicate that different SUMO paralogues determine the fates of WT and mutant CFTRs, and they suggest that a paralogue switch during biogenesis can direct these proteins to different outcomes: biogenesis versus degradation.
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Regulador de Condutância Transmembrana em Fibrose Cística/biossíntese , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteínas Mutantes/biossíntese , Proteínas Mutantes/metabolismo , Proteólise , Homologia de Sequência de Aminoácidos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Brônquios/patologia , Linhagem Celular , Membrana Celular/metabolismo , Fibrose Cística/patologia , Retículo Endoplasmático/metabolismo , Células Epiteliais/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Estabilidade Proteica , Sumoilação , Fatores de Transcrição/metabolismo , UbiquitinaçãoRESUMO
The protein chaperones heat shock protein 70 (Hsp70) and Hsp90 are required for de novo folding of proteins and protect against misfolding-related cellular stresses by directing misfolded or slowly folding proteins to the ubiquitin/proteasome system (UPS) or autophagy/lysosomal degradation pathways. Here, we examined the role of the Bcl2-associated athanogene (BAG) family of Hsp70-specific nucleotide-exchange factors in the biogenesis and functional correction of genetic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) whose mutations cause cystic fibrosis (CF). We show that siRNA-mediated silencing of BAG1 and -3, two BAG members linked to the clearance of misfolded proteins via the UPS and autophagy pathways, respectively, leads to functional correction of F508del-CFTR and other disease-associated CFTR variants. BAG3 silencing was the most effective, leading to improved F508del-CFTR stability, trafficking, and restoration of cell-surface function, both alone and in combination with the FDA-approved CFTR corrector, VX-809. We also found that the BAG3 silencing-mediated correction of F508del-CFTR restores the autophagy pathway, which is defective in F508del-CFTR-expressing cells, likely because of the maladaptive stress response in CF pathophysiology. These results highlight the potential therapeutic benefits of targeting the cellular chaperone system to improve the functional folding of CFTR variants contributing to CF and possibly other protein-misfolding-associated diseases.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Proteínas de Choque Térmico HSP70/metabolismo , Mutação , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Estabilidade Proteica , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno/genética , Regulação para CimaRESUMO
Several members of the SLC26A family of anion transporters associate with CFTR, forming complexes in which CFTR and SLC26A functions are reciprocally regulated. These associations are thought to be facilitated by PDZ scaffolding interactions. CFTR has been shown to be positively regulated by NHERF-1, and negatively regulated by CAL in airway epithelia. However, it is unclear which PDZ-domain protein(s) interact with SLC26A9, a SLC26A family member found in airway epithelia. We have previously shown that primary, human bronchial epithelia (HBE) from non-CF donors exhibit constitutive anion secretion attributable to SLC26A9. However, constitutive anion secretion is absent in HBE from CF donors. We examined whether changes in SLC26A9 constitutive activity could be attributed to a loss of CFTR trafficking, and what role PDZ interactions played. HEK293 coexpressing SLC26A9 with the trafficking mutant F508del CFTR exhibited a significant reduction in constitutive current compared with cells coexpressing SLC26A9 and wt CFTR. We found that SLC26A9 exhibits complex glycosylation when coexpressed with F508del CFTR, but its expression at the plasma membrane is decreased. SLC26A9 interacted with both NHERF-1 and CAL, and its interaction with both significantly increased with coexpression of wt CFTR. However, coexpression with F508del CFTR only increased SLC26A9's interaction with CAL. Mutation of SLC26A9's PDZ motif decreased this association with CAL, and restored its constitutive activity. Correcting aberrant F508del CFTR trafficking in CF HBE with corrector VX-809 also restored SLC26A9 activity. We conclude that when SLC26A9 is coexpressed with F508del CFTR, its trafficking defect leads to a PDZ motif-sensitive intracellular retention of SLC26A9.
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Antiporters/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Mutação/genética , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Antiporters/química , Proteínas de Transporte , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Células Epiteliais/metabolismo , Imunofluorescência , Proteínas da Matriz do Complexo de Golgi , Células HEK293 , Humanos , Imunoprecipitação , Proteínas de Membrana , Proteínas de Membrana Transportadoras , Modelos Biológicos , Domínios PDZ , Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Transportadores de SulfatoRESUMO
In the shark rectal gland (SRG), apical chloride secretion through CFTR channels is electrically coupled to a basolateral K+ conductance whose type and molecular identity are unknown. We performed studies in the perfused SRG with 17 K+ channel inhibitors to begin this search. Maximal chloride secretion was markedly inhibited by low-perfusate pH, bupivicaine, anandamide, zinc, quinidine, and quinine, consistent with the properties of an acid-sensitive, four-transmembrane, two-pore-domain K+ channel (4TM-K2P). Using PCR with degenerate primers to this family, we identified a TASK-1 fragment in shark rectal gland, brain, gill, and kidney. Using 5' and 3' rapid amplification of cDNA ends PCR and genomic walking, we cloned the full-length shark gene (1,282 bp), whose open reading frame encodes a protein of 375 amino acids that was 80% identical to the human TASK-1 protein. We expressed shark and human TASK-1 cRNA in Xenopus oocytes and characterized these channels using two-electrode voltage clamping. Both channels had identical current-voltage relationships (outward rectifying) and a reversal potential of -90 mV. Both were inhibited by quinine, bupivicaine, and acidic pH. The pKa for current inhibition was 7.75 for shark TASK-1 vs. 7.37 for human TASK-1, values similar to the arterial pH for each species. We identified this protein in SRG by Western blot and confocal immunofluorescent microscopy and detected the protein in SRG and human airway cells. Shark TASK-1 is the major K+ channel coupled to chloride secretion in the SRG, is the oldest 4TM 2P family member identified, and is the first TASK-1 channel identified to play a role in setting the driving force for chloride secretion in epithelia. The detection of this potassium channel in mammalian lung tissue has implications for human biology and disease.
Assuntos
Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio/metabolismo , Glândula de Sal/metabolismo , Tubarões/metabolismo , Sequência de Aminoácidos , Animais , DNA Complementar/genética , Cação (Peixe)/metabolismo , Humanos , Proteínas do Tecido Nervoso/genética , Oócitos/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Xenopus laevis/genéticaRESUMO
Unesterified cholesterol accumulates in late endosomes in cells expressing the misfolded cystic fibrosis transmembrane conductance regulator (CFTR). CFTR misfolding in the endoplasmic reticulum (ER) or general activation of ER stress led to dynein-mediated clustering of cholesterol-loaded late endosomes at the Golgi region, a process regulated by ER-localized VAMP-associated proteins (VAPs). We hypothesized that VAPs serve as intracellular receptors that couple lipid homeostasis through interactions with two phenylalanines in an acidic track (FFAT) binding signals (found in lipid sorting and sensing proteins, LSS) with proteostasis regulation. VAPB inhibited the degradation of ΔF508-CFTR. The activity was mapped to the ligand-binding major sperm protein (MSP) domain, which was sufficient in regulating CFTR biogenesis. We identified mutations in an unstructured loop within the MSP that uncoupled VAPB-regulated CFTR biogenesis from basic interactions with FFAT. Using this information, we defined functional and physical interactions between VAPB and proteostasis regulators (ligands), including the unfolded protein response sensor ATF6 and the ER degradation cluster that included FAF1, VCP, BAP31, and Derlin-1. VAPB inhibited the degradation of ΔF508-CFTR in the ER through interactions with the RMA1-Derlin-BAP31-VCP pathway. Analysis of pseudoligands containing tandem FFAT signals supports a competitive model for VAP interactions that direct CFTR biogenesis. The results suggest a model in which VAP-ligand binding couples proteostasis and lipid homeostasis leading to observed phenotypes of lipid abnormalities in protein folding diseases.
Assuntos
Colesterol/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteólise , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Adenosina Trifosfatases , Proteínas Reguladoras de Apoptose , Sítios de Ligação , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Células HeLa , Homeostase , Humanos , Proteínas de Membrana/metabolismo , Ligação Proteica , Estabilidade Proteica , Ubiquitina-Proteína Ligases/metabolismo , Proteína com ValosinaRESUMO
More than 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been described that confer a range of molecular cell biological and functional phenotypes. Most of these mutations lead to compromised anion conductance at the apical plasma membrane of secretory epithelia and cause cystic fibrosis (CF) with variable disease severity. Based on the molecular phenotypic complexity of CFTR mutants and their susceptibility to pharmacotherapy, it has been recognized that mutations may impose combinatorial defects in CFTR channel biology. This notion led to the conclusion that the combination of pharmacotherapies addressing single defects (e.g., transcription, translation, folding, and/or gating) may show improved clinical benefit over available low-efficacy monotherapies. Indeed, recent phase 3 clinical trials combining ivacaftor (a gating potentiator) and lumacaftor (a folding corrector) have proven efficacious in CF patients harboring the most common mutation (deletion of residue F508, ΔF508, or Phe508del). This drug combination was recently approved by the U.S. Food and Drug Administration for patients homozygous for ΔF508. Emerging studies of the structural, cell biological, and functional defects caused by rare mutations provide a new framework that reveals a mixture of deficiencies in different CFTR alleles. Establishment of a set of combinatorial categories of the previously defined basic defects in CF alleles will aid the design of even more efficacious therapeutic interventions for CF patients.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Animais , Agonistas dos Canais de Cloreto/farmacologia , Agonistas dos Canais de Cloreto/uso terapêutico , Fibrose Cística/classificação , Fibrose Cística/tratamento farmacológico , Regulador de Condutância Transmembrana em Fibrose Cística/agonistas , Predisposição Genética para Doença , Humanos , Ativação do Canal Iônico , Mutação de Sentido IncorretoRESUMO
Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians. It is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, which encodes an apical membrane anion channel that is required for regulating the volume and composition of epithelial secretions. The most common CFTR mutation, present on at least one allele in >90% of CF patients, deletes phenylalanine at position 508 (F508del), which causes the protein to misfold. Endoplasmic reticulum (ER) quality control elicits the degradation of mutant CFTR, compromising its trafficking to the epithelial cell apical membrane. The absence of functional CFTR leads to depletion of airway surface liquid, impaired clearance of mucus and bacteria from the lung, and predisposes to recurrent infections. Ultimately, respiratory failure results from inflammation and bronchiectasis. Although high throughput screening has identified small molecules that can restore the anion transport function of F508del CFTR, they correct less than 15% of WT CFTR activity, yielding insufficient clinical benefit. To date, most primary CF drug discovery assays have employed measurements of CFTR's anion transport function, a method that depends on the recruitment of a functional CFTR to the cell surface, involves multiple wash steps, and relies on a signal that saturates rapidly. Screening efforts have also included assays for detection of extracellularly HA-tagged or HRP-tagged CFTR, which require multiple washing steps. We have recently developed tools and cell lines that report the correction of mutant CFTR trafficking by currently available small molecules, and have extended this assay to the 96-well format. This new and simple no-wash assay of F508del CFTR at the cell surface may permit the discovery of more efficacious drugs, and hopefully thereby prevent the catastrophic effects of this disease. In addition, the modular design of this platform should make it useful for other diseases where loss-of-function results from folding and/or trafficking defects in membrane proteins.
Assuntos
Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/agonistas , Ensaios de Triagem em Larga Escala , Proteínas Recombinantes de Fusão/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Corantes Fluorescentes/química , Expressão Gênica , Células HEK293 , Humanos , Imagem Molecular , Mutação , Plasmídeos/química , Plasmídeos/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/metabolismo , Corantes de Rosanilina/química , Anticorpos de Cadeia Única/química , TransfecçãoRESUMO
A newly identified pathway for selective degradation of the common mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), F508del, is initiated by binding of the small heat shock protein, Hsp27. Hsp27 collaborates with Ubc9, the E2 enzyme for protein SUMOylation, to selectively degrade F508del CFTR via the SUMO-targeted ubiquitin E3 ligase, RNF4 (RING finger protein 4) (1). Here, we ask what properties of CFTR are sensed by the Hsp27-Ubc9 pathway by examining the ability of NBD1 (locus of the F508del mutation) to mimic the disposal of full-length (FL) CFTR. Similar to FL CFTR, F508del NBD1 expression was reduced 50-60% by Hsp27; it interacted preferentially with the mutant and was modified primarily by SUMO-2. Mutation of the consensus SUMOylation site, Lys(447), obviated Hsp27-mediated F508del NBD1 SUMOylation and degradation. As for FL CFTR and NBD1 in vivo, SUMO modification using purified components in vitro was greater for F508del NBD1 versus WT and for the SUMO-2 paralog. Several findings indicated that Hsp27-Ubc9 targets the SUMOylation of a transitional, non-native conformation of F508del NBD1: (a) its modification decreased as [ATP] increased, reflecting stabilization of the nucleotide-binding domain by ligand binding; (b) a temperature-induced increase in intrinsic fluorescence, which reflects formation of a transitional NBD1 conformation, was followed by its SUMO modification; and (c) introduction of solubilizing or revertant mutations to stabilize F508del NBD1 reduced its SUMO modification. These findings indicate that the Hsp27-Ubc9 pathway recognizes a non-native conformation of mutant NBD1, which leads to its SUMO-2 conjugation and degradation by the ubiquitin-proteasome system.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Proteínas de Choque Térmico HSP27/genética , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Proteólise , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , SumoilaçãoRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is generally responsible for the cAMP/PKA regulated anion conductance at the apical membranes of secretory epithelial cells. Mutations in CFTR underlie cystic fibrosis (CF), in which the most common variant, F508del, causes protein misfolding and its proteasome-mediated degradation. A new pathway that contributes to mutant CFTR degradation is mediated by the small heat shock protein, Hsp27, which cooperates with Ubc9, the E2 enzyme for SUMOylation, to selectively conjugate mutant CFTR with SUMO-2/3. This SUMO paralog can form polychains, which are recognized by the ubiquitin E3 enzyme, RNF4, leading to CFTR ubiquitylation and recognition by the proteasome. We found also that F508del CFTR could be modified by SUMO-1, a paralog that does not support SUMO polychain formation. The use of different SUMO paralogs to modify and target a single substrate for divergent purposes is not uncommon. In this short review we discuss the possibility that conjugation with SUMO-1 could protect mutant CFTR from disposal by RNF4 and similar ubiquitin ligases. We hypothesize that such a pathway could contribute to therapeutic efforts to stabilize immature mutant CFTR and thereby enhance the action of therapeutics that correct CFTR trafficking to the apical membranes.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Mucosa Respiratória/metabolismo , Proteína SUMO-1/metabolismo , Sumoilação , Animais , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Predisposição Genética para Doença , Humanos , Mutação , Proteínas Nucleares/metabolismo , Fenótipo , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Proteólise , Fatores de Transcrição/metabolismoRESUMO
The most prevalent form of cystic fibrosis arises from an amino acid deletion in the cystic fibrosis transmembrane conductance regulator, CFTR. A recently approved treatment for individuals homozygous for this mutation combines a chemical corrector, which helps CFTR fold, and a potentiator that increases CFTR channel activity.
Assuntos
Aminofenóis/uso terapêutico , Aminopiridinas/uso terapêutico , Benzodioxóis/uso terapêutico , Fibrose Cística/tratamento farmacológico , Quinolonas/uso terapêutico , Fibrose Cística/genética , Fibrose Cística/história , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Combinação de Medicamentos , Deleção de Genes , História do Século XX , História do Século XXI , HumanosRESUMO
Cystic fibrosis (CF) is a lethal genetic disease caused by the loss or dysfunction of the CF transmembrane conductance regulator (CFTR) channel. F508del is the most prevalent mutation of the CFTR gene and encodes a protein defective in folding and processing. VX-809 has been reported to facilitate the folding and trafficking of F508del-CFTR and augment its channel function. The mechanism of action of VX-809 has been poorly understood. In this study, we sought to answer a fundamental question underlying the mechanism of VX-809: does it bind CFTR directly in order to exert its action? We synthesized two VX-809 derivatives, ALK-809 and SUL-809, that possess an alkyne group and retain the rescue capacity of VX-809. By using Cu(I) -catalyzed click chemistry, we provide evidence that the VX-809 derivatives bind CFTR directly in vitro and in cells. Our findings will contribute to the elucidation of the mechanism of action of CFTR correctors and the design of more potent therapeutics to combat CF.