RESUMO
Understanding the mechanism of action of modulator compounds for the cystic fibrosis transmembrane conductance regulator (CFTR) is key for the optimization of therapeutics as well as obtaining insights into the molecular mechanisms of CFTR function. We demonstrate the direct binding of VX-809 to the first nucleotide-binding domain (NBD1) of human CFTR. Disruption of the interaction between C-terminal helices and the NBD1 core upon VX-809 binding is observed from chemical shift changes in the NMR spectra of residues in the helices and on the surface of ß-strands S3, S9, and S10. Binding to VX-809 leads to a significant negative shift in NBD1 thermal melting temperature (Tm), pointing to direct VX-809 interaction shifting the NBD1 conformational equilibrium. An inter-residue correlation analysis of the chemical shift changes provides evidence of allosteric coupling between the direct binding site and the NBD1:CL4 interface, thus enabling effects on the interface in the absence of direct binding in that location. These NMR binding data and the negative Tm shifts are very similar to those previously reported by us for binding of the dual corrector-potentiator CFFT-001 to NBD1 (Hudson et al., 2012), suggesting that the two compounds may share some aspects of their mechanisms of action. Although previous studies have shown an important role for VX-809 in modulating the conformation of the first membrane spanning domain (Aleksandrov et al., 2012; Ren et al., 2013), this additional mode of VX-809 binding provides insight into conformational dynamics and allostery within CFTR.
Assuntos
Aminopiridinas/metabolismo , Benzodioxóis/metabolismo , Proteínas de Transporte/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulação Alostérica/fisiologia , Aminopiridinas/química , Benzodioxóis/química , Sítios de Ligação/fisiologia , Proteínas de Transporte/química , Regulador de Condutância Transmembrana em Fibrose Cística/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Early-onset torsion dystonia (EOTD) is a neurological disorder characterized by involuntary and sustained muscle contractions that can lead to paralysis and abnormal posture. EOTD is associated with the deletion of a glutamate (ΔE) in torsinA, an endoplasmic reticulum (ER) resident AAA(+) ATPase. To date, the effect of ΔE on torsinA and the reason that this mutation results in EOTD are unclear. Moreover, there are no specific therapeutic options to treat EOTD. To define the underlying biochemical defects associated with torsinAΔE and to uncover factors that might be targeted to offset defects associated with torsinAΔE, we developed a yeast torsinA expression system and tested the roles of ER chaperones in mediating the folding and stability of torsinA and torsinAΔE. We discovered that the ER lumenal Hsp70, BiP, an associated Hsp40, Scj1, and a nucleotide exchange factor, Lhs1, stabilize torsinA and torsinAΔE. BiP also maintained torsinA and torsinAΔE solubility. Mutations predicted to compromise specific torsinA functional motifs showed a synthetic interaction with the ΔE mutation and destabilized torsinAΔE, suggesting that the ΔE mutation predisposes torsinA to defects in the presence of secondary insults. In this case, BiP was required for torsinAΔE degradation, consistent with data that specific chaperones exhibit either pro-degradative or pro-folding activities. Finally, using two independent approaches, we established that BiP stabilizes torsinA and torsinAΔE in mammalian cells. Together, these data define BiP as the first identified torsinA chaperone, and treatments that modulate BiP might improve symptoms associated with EOTD.
Assuntos
Adenosina Trifosfatases/metabolismo , Distonia Muscular Deformante/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Animais , Western Blotting , Linhagem Celular , Distonia Muscular Deformante/genética , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Glicosilação , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Células HeLa , Proteínas de Choque Térmico/genética , Humanos , Microscopia de Fluorescência , Chaperonas Moleculares/genética , Mutação , Estabilidade Proteica , Interferência de RNA , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: In 2017, the US Food and Drug Administration initiated expansion of drug labels for the treatment of cystic fibrosis (CF) to include CF transmembrane conductance regulator (CFTR) gene variants based on in vitro functional studies. This study aims to identify CFTR variants that result in increased chloride (Cl-) transport function by the CFTR protein after treatment with the CFTR modulator combination elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA). These data may benefit people with CF (pwCF) who are not currently eligible for modulator therapies. METHODS: Plasmid DNA encoding 655 CFTR variants and wild-type (WT) CFTR were transfected into Fisher Rat Thyroid cells that do not natively express CFTR. After 24 h of incubation with control or TEZ and ELX, and acute addition of IVA, CFTR function was assessed using the transepithelial current clamp conductance assay. Each variant's forskolin/cAMP-induced baseline Cl- transport activity, responsiveness to IVA alone, and responsiveness to the TEZ/ELX/IVA combination were measured in three different laboratories. Western blots were conducted to evaluate CFTR protein maturation and complement the functional data. RESULTS AND CONCLUSIONS: 253 variants not currently approved for CFTR modulator therapy showed low baseline activity (<10 % of normal CFTR Cl- transport activity). For 152 of these variants, treatment with ELX/TEZ/IVA improved the Cl- transport activity by ≥10 % of normal CFTR function, which is suggestive of clinical benefit. ELX/TEZ/IVA increased CFTR function by ≥10 percentage points for an additional 140 unapproved variants with ≥10 % but <50 % of normal CFTR function at baseline. These findings significantly expand the number of rare CFTR variants for which ELX/TEZ/IVA treatment should result in clinical benefit.
Assuntos
Aminofenóis , Benzodioxóis , Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Indóis , Pirazóis , Quinolonas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Quinolonas/uso terapêutico , Quinolonas/farmacologia , Aminofenóis/uso terapêutico , Aminofenóis/farmacologia , Animais , Ratos , Humanos , Benzodioxóis/uso terapêutico , Benzodioxóis/farmacologia , Indóis/uso terapêutico , Indóis/farmacologia , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Combinação de Medicamentos , Agonistas dos Canais de Cloreto/uso terapêutico , Pirrolidinas/farmacologia , Pirrolidinas/uso terapêuticoRESUMO
Early onset dystonia is commonly associated with the deletion of one of a pair of glutamate residues (ΔE302/303) near the C terminus of torsinA, a member of the AAA+ protein family (ATPases associated with a variety of cellular activities) located in the endoplasmic reticulum lumen. The functional consequences of the disease-causing mutation, ΔE, are not currently understood. By contrast to other AAA+ proteins, torsin proteins contain two conserved cysteine residues in the C-terminal domain, one of which is located in the nucleotide sensor II motif. Depending on redox status, an ATP hydrolysis mutant of torsinA interacts with lamina-associated polypeptide 1 (LAP1) and lumenal domain like LAP1 (LULL1). Substitution of the cysteine in sensor II diminishes the redox-regulated interaction of torsinA with these substrates. Significantly, the dystonia-causing mutation, ΔE, alters the ability of torsinA to mediate the redox-regulated interactions with LAP1 and LULL1. Limited proteolysis experiments reveal redox- and mutation-dependent changes in the local conformation of torsinA as a function of nucleotide binding. These results indicate that the cysteine-containing sensor II plays a critical role in redox sensing and the nucleotide and partner binding functions of torsinA and suggest that loss of this function of torsinA contributes to the development of DYT1 dystonia.
Assuntos
Trifosfato de Adenosina/metabolismo , Distonia/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Motivos de Aminoácidos , Animais , Células COS , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Chlorocebus aethiops , Distonia/genética , Proteínas de Choque Térmico HSC70/genética , Proteínas de Choque Térmico HSC70/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/genética , Mutação , Oxirredução , Ligação Proteica , Conformação ProteicaRESUMO
The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations. However, the specific steps of folding and assembly of full-length cystic fibrosis transmembrane conductance regulator (CFTR) directly altered by the disease-causing mutation are unclear. To elucidate the effects of the ΔF508 mutation, on various steps in CFTR folding, a series of misfolding and suppressor mutations in the nucleotide binding and transmembrane domains were evaluated for effects on the folding and maturation of the protein. The results indicate that the isolated NBD1 responds to both the ΔF508 mutation and intradomain suppressors of this mutation. In addition, identification of a novel second site suppressor of the defect within the second transmembrane domain suggests that ΔF508 also effects interdomain interactions critical for later steps in the biosynthesis of CFTR.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Mutação , Fenilalanina/genética , Sítios de Ligação/genética , Western Blotting , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Fenilalanina/química , Fenilalanina/metabolismo , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Deleção de Sequência , Supressão GenéticaRESUMO
Chloride absorption and bicarbonate secretion are vital functions of epithelia, as highlighted by cystic fibrosis and diseases associated with mutations in members of the SLC26 chloride-bicarbonate exchangers. Many SLC26 transporters (SLC26T) are expressed in the luminal membrane together with CFTR, which activates electrogenic chloride-bicarbonate exchange by SLC26T. However, the ability of SLC26T to regulate CFTR and the molecular mechanism of their interaction are not known. We report here a reciprocal regulatory interaction between the SLC26T DRA, SLC26A6 and CFTR. DRA markedly activates CFTR by increasing its overall open probablity (NP(o)) sixfold. Activation of CFTR by DRA was facilitated by their PDZ ligands and binding of the SLC26T STAS domain to the CFTR R domain. Binding of the STAS and R domains is regulated by PKA-mediated phosphorylation of the R domain. Notably, CFTR and SLC26T co-localize in the luminal membrane and recombinant STAS domain activates CFTR in native duct cells. These findings provide a new understanding of epithelial chloride and bicarbonate transport and may have important implications for both cystic fibrosis and diseases associated with SLC26T.
Assuntos
Antiporters , Bicarbonatos/metabolismo , Proteínas de Transporte/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Ativação do Canal Iônico/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Proteínas de Transporte/genética , Linhagem Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Cloretos/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Ativação do Canal Iônico/genética , Ligantes , Proteínas de Membrana/genética , Fosforilação , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Transportadores de Sulfato , Equilíbrio Hidroeletrolítico/genéticaRESUMO
Allelic heterogeneity in disease-causing genes presents a substantial challenge to the translation of genomic variation into clinical practice. Few of the almost 2,000 variants in the cystic fibrosis transmembrane conductance regulator gene CFTR have empirical evidence that they cause cystic fibrosis. To address this gap, we collected both genotype and phenotype data for 39,696 individuals with cystic fibrosis in registries and clinics in North America and Europe. In these individuals, 159 CFTR variants had an allele frequency of l0.01%. These variants were evaluated for both clinical severity and functional consequence, with 127 (80%) meeting both clinical and functional criteria consistent with disease. Assessment of disease penetrance in 2,188 fathers of individuals with cystic fibrosis enabled assignment of 12 of the remaining 32 variants as neutral, whereas the other 20 variants remained of indeterminate effect. This study illustrates that sourcing data directly from well-phenotyped subjects can address the gap in our ability to interpret clinically relevant genomic variation.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , FenótipoRESUMO
Many missense mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) result in its misfolding, endoplasmic reticulum (ER) accumulation, and, thus, cystic fibrosis. A number of these mutations are located in the predicted CFTR transmembrane (TM) spans and have been projected to alter span integration. However, the boundaries of the spans have not been precisely defined experimentally. In this study, the ER luminal integration profiles of TM1 and TM2 were determined using the ER glycosylation machinery, and the effects of the CF-causing mutations G85E and G91R thereon were assessed. The mutations either destabilize the integrated conformation or alter the TM1 ER integration profile. G85E misfolding is based in TM1 destabilization by glutamic acid and loss of glycine and correlates with the temperature-insensitive ER accumulation of immature full-length CFTR harboring the mutation. By contrast, temperature-dependent misfolding owing to the G91R mutation depends on the introduction of the basic side chain rather than the loss of the glycine. This work demonstrates that CF-causing mutations predicted to have similar effects on CFTR structure actually result in disparate molecular perturbations that underlie ER accumulation and the pathology of CF.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/genética , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Ácido Glutâmico/química , Glicina/química , Glicosilação , Humanos , Mutação , Dobramento de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , TemperaturaRESUMO
ATP-binding cassette (ABC) transporters harvest the energy present in cellular ATP to drive the translocation of a structurally diverse set of solutes across the membrane barriers of eubacteria, archaebacteria, and eukaryotes. The positively cooperative ATPase activity (Hill coefficient, 1.7) of a model soluble cassette of known structure, MJ0796, from Methanococcus jannaschii indicates that at least two binding sites participate in the catalytic reaction. Mutation of the catalytic base in MJ0796, E171Q, produced a cassette that can bind but not efficiently hydrolyze ATP. The equivalent mutation (E179Q) in a homologous cassette, MJ1267, had an identical effect. Both mutant cassettes formed dimers in the presence of ATP but not ADP, indicating that the energy of ATP binding is first coupled to the transport cycle through a domain association reaction. The non-hydrolyzable nucleotides adenosine 5'-(beta,gamma-imino)triphosphate and adenosine 5'-3-O-(thio)triphosphate were poor analogues of ATP in terms of their ability to promote dimerization. Moreover, inclusion of MgCl2, substitution of KCl for NaCl, or alterations in the polarity of the side chain at the catalytic base all weakened the ATP-dependent dimer, suggesting that electrostatic interactions are critical for the association reaction. Thus, upon hydrolysis of bound ATP and the release of product, both electrostatic and conformational changes drive the cassettes apart, providing a second opportunity to couple free energy changes to the transport reaction.
Assuntos
Trifosfato de Adenosina/metabolismo , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Cromatografia em Gel , Dimerização , Relação Dose-Resposta a Droga , Hidrólise , Cinética , Cloreto de Magnésio/farmacologia , Mathanococcus/química , Mathanococcus/genética , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Cloreto de Potássio/farmacologia , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , TermodinâmicaRESUMO
It has been proposed that the reaction cycle of ATP binding cassette (ABC) transporters is driven by dimerization of their ABC motor domains upon binding ATP at their mutual interface. However, no such ATP sandwich complex has been observed for an ABC from an ABC transporter. In this paper, we report the crystal structure of a stable dimer formed by the E171Q mutant of the MJ0796 ABC, which is hydrolytically inactive due to mutation of the catalytic base. The structure shows a symmetrical dimer in which two ATP molecules are each sandwiched between the Walker A motif in one subunit and the LSGGQ signature motif in the other subunit. These results establish the stereochemical basis of the power stroke of ABC transporter pumps.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Difosfato de Adenosina/metabolismo , Regulação Alostérica , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Polarização de Fluorescência , Mathanococcus/química , Modelos Moleculares , Proteínas Motores Moleculares/genética , Dados de Sequência Molecular , Mutação , Conformação Proteica , Estrutura Terciária de Proteína , Sódio/metabolismo , Eletricidade EstáticaRESUMO
PA700, the 19 S regulatory complex of the 26 S proteasome, plays a central role in the recognition and efficient degradation of misfolded proteins. PA700 promotes degradation by recruiting proteasomal substrates utilizing polyubiquitin chains and chaperone-like binding activities and by opening the access to the core of the 20 S proteasome to promote degradation. Here we provide evidence that PA700 in addition to binding misfolded protein substrates also acts to remodel their conformation prior to proteolysis. Scrambled RNase A (scRNase A), a misfolded protein, only slowly refolds spontaneously into an active form because of the rate-limiting unfolding of misfolded disulfide isomers. Notably, PA700 accelerates the rate of reactivation of scRNase A, consistent with its ability to increase the exposure of these disulfide bonds to the solvent. In this regard, PA700 also exposes otherwise buried sites to digestion by exogenous chymotrypsin in a polyubiquitinated enzymatically active substrate, pentaubiquitinated dihydrofolate reductase, Ub(5)DHFR. The dihydrofolate reductase ligand methotrexate counters the ability of PA700 to promote digestion by chymotrypsin. Together, these results indicate that in addition to increasing substrate affinity and opening the access channel to the catalytic sites, PA700 activates proteasomal degradation by remodeling the conformation of protein substrates.