RESUMEN
Misfolded proteins are often cytotoxic, unless cellular systems prevent their accumulation. Data presented here uncover a mechanism by which defects in secretory proteins lead to a dramatic reduction in their mRNAs and protein expression. When mutant signal sequences fail to bind to the signal recognition particle (SRP) at the ribosome exit site, the nascent chain instead contacts Argonaute2 (Ago2), and the mutant mRNAs are specifically degraded. Severity of signal sequence mutations correlated with increased proximity of Ago2 to nascent chain and mRNA degradation. Ago2 knockdown inhibited degradation of the mutant mRNA, while overexpression of Ago2 or knockdown of SRP54 promoted degradation of secretory protein mRNA. The results reveal a previously unappreciated general mechanism of translational quality control, in which specific mRNA degradation preemptively regulates aberrant protein production (RAPP).
Asunto(s)
Biosíntesis de Proteínas , Pliegue de Proteína , Estabilidad del ARN , Partícula de Reconocimiento de Señal/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Argonautas/metabolismo , Perros , Células HeLa , Humanos , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de SecuenciaRESUMEN
Misfolding of ΔF508 cystic fibrosis (CF) transmembrane conductance regulator (CFTR) underlies pathology in most CF patients. F508 resides in the first nucleotide-binding domain (NBD1) of CFTR near a predicted interface with the fourth intracellular loop (ICL4). Efforts to identify small molecules that restore function by correcting the folding defect have revealed an apparent efficacy ceiling. To understand the mechanistic basis of this obstacle, positions statistically coupled to 508, in evolved sequences, were identified and assessed for their impact on both NBD1 and CFTR folding. The results indicate that both NBD1 folding and interaction with ICL4 are altered by the ΔF508 mutation and that correction of either individual process is only partially effective. By contrast, combination of mutations that counteract both defects restores ΔF508 maturation and function to wild-type levels. These results provide a mechanistic rationale for the limited efficacy of extant corrector compounds and suggest approaches for identifying compounds that correct both defective steps.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Supresión Genética , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Fibrosis Quística/genética , Fibrosis Quística/terapia , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Ratones , Modelos Moleculares , Pliegue de Proteína , Estructura Terciaria de ProteínaRESUMEN
While primary cystic fibrosis (CF) and non-CF human bronchial epithelial basal cells (HBECs) accurately represent in vivo phenotypes, one barrier to their wider use has been a limited ability to clone and expand cells in sufficient numbers to produce rare genotypes using genome-editing tools. Recently, conditional reprogramming of cells (CRC) with a Rho-associated protein kinase (ROCK) inhibitor and culture on an irradiated fibroblast feeder layer resulted in extension of the life span of HBECs, but differentiation capacity and CF transmembrane conductance regulator (CFTR) function decreased as a function of passage. This report details modifications to the standard HBEC CRC protocol (Mod CRC), including the use of bronchial epithelial cell growth medium, instead of F medium, and 2% O2, instead of 21% O2, that extend HBEC life span while preserving multipotent differentiation capacity and CFTR function. Critically, Mod CRC conditions support clonal growth of primary HBECs from a single cell, and the resulting clonal HBEC population maintains multipotent differentiation capacity, including CFTR function, permitting gene editing of these cells. As a proof-of-concept, CRISPR/Cas9 genome editing and cloning were used to introduce insertions/deletions in CFTR exon 11. Mod CRC conditions overcome many barriers to the expanded use of HBECs for basic research and drug screens. Importantly, Mod CRC conditions support the creation of isogenic cell lines in which CFTR is mutant or wild-type in the same genetic background with no history of CF to enable determination of the primary defects of mutant CFTR.
Asunto(s)
Bronquios/metabolismo , Diferenciación Celular , Fibrosis Quística/metabolismo , Células Madre Multipotentes/metabolismo , Células 3T3 , Animales , Bronquios/patología , Sistemas CRISPR-Cas , Técnicas de Cultivo de Célula , Células Cultivadas , Técnicas de Reprogramación Celular , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Edición Génica , Humanos , Ratones , Células Madre Multipotentes/patología , Factores de TiempoRESUMEN
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.
Asunto(s)
Aminopiridinas/metabolismo , Benzodioxoles/metabolismo , Proteínas Portadoras/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulación Alostérica/fisiología , Aminopiridinas/química , Benzodioxoles/química , Sitios de Unión/fisiología , Proteínas Portadoras/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Humanos , Péptidos y Proteínas de Señalización Intracelular , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Females have a more severe clinical course than males in terms of several inflammatory lung conditions. Notably, females with cystic fibrosis (CF) suffer worse outcomes, particularly in the setting of Pseudomonas aeruginosa infection. Sex hormones have been implicated in experimental and clinical studies; however, immune mechanisms responsible for this sex-based disparity are unknown and the specific sex hormone target for therapeutic manipulation has not been identified. The objective of this study was to assess mechanisms behind the impact of female sex hormones on host immune responses to P. aeruginosa We used wild-type and CF mice, which we hormone manipulated, inoculated with P. aeruginosa, and then examined for outcomes and inflammatory responses. Neutrophils isolated from mice and human subjects were tested for responses to P. aeruginosa We found that female mice inoculated with P. aeruginosa died earlier and showed slower bacterial clearance than males (P < 0.0001). Ovariectomized females supplemented with 17ß-estradiol succumbed to P. aeruginosa challenge earlier than progesterone- or vehicle-supplemented mice (P = 0.0003). 17ß-Estradiol-treated ovariectomized female mice demonstrated increased lung levels of inflammatory cytokines, and when rendered neutropenic the mortality difference was abrogated. Neutrophils treated with 17ß-estradiol demonstrated an enhanced oxidative burst but decreased P. aeruginosa killing and earlier cell necrosis. The estrogen receptor (ER) antagonist ICI 182,780 improved survival in female mice infected with P. aeruginosa and restored neutrophil function. We concluded that ER antagonism rescues estrogen-mediated neutrophil dysfunction and improves survival in response to P. aeruginosa ER-mediated processes may explain the sex-based mortality gap in CF and other inflammatory lung illnesses, and the ER blockade represents a rational therapeutic strategy.
Asunto(s)
Estradiol/farmacología , Inmunidad Innata/efectos de los fármacos , Neutrófilos/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Receptores de Estrógenos/antagonistas & inhibidores , Infecciones del Sistema Respiratorio/inmunología , Animales , Fibrosis Quística/microbiología , Estradiol/administración & dosificación , Estrógenos/administración & dosificación , Estrógenos/sangre , Estrógenos/farmacología , Femenino , Humanos , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Masculino , Ratones , Necrosis , Neutropenia/inmunología , Neutropenia/microbiología , Neutrófilos/efectos de los fármacos , Neutrófilos/microbiología , Ovariectomía , Progesterona/administración & dosificación , Progesterona/sangre , Infecciones por Pseudomonas/microbiología , Estallido Respiratorio , Infecciones del Sistema Respiratorio/microbiologíaRESUMEN
BACKGROUND: Semi-synthetic oleanane triterpenoid antioxidant inflammation modulators (tpAIMs) are small molecules that interact with KEAP1 cysteine residue 151 (C151) and activate NRF2. Exploration of the structure-activity relationship between the tpAIMs and KEAP1 is limited by the predominantly hydrocarbon nature of the oleanane triterpenoid pentacyclic ring structure. Therefore, we used novel, chemically-tractable, synthetic antioxidant inflammation modulators (sAIMs) to probe the stereoselectivity of the ligand-protein interaction. METHODS: We measured several parameters of NRF2 activation to assess the potency of sAIM enantiomers with natural (tpAIM-like) 4(S),5(S),10(R) or unnatural 4(R),5(R),10(S) configurations. Additionally, we determined the crystal structure of the KEAP1 BTB domain in complex with two different sAIMs. RESULTS: We found that the potencies of sAIM enantiomers in the natural configuration were similar to those of the tpAIM, RTA 405. Strikingly, sAIM enantiomers in the unnatural configuration were 10- to 40-fold less potent than their natural counterparts. Crystallographic studies of sAIMs in complex with the KEAP1 BTB domain demonstrated that these ligands form a covalent bond with C151 and revealed the presence of additional hydrogen bonds, Van der Waals interactions, and pi-stacking interactions. CONCLUSIONS: Although KEAP1 C151 is required for NRF2 activation by tpAIMs and sAIMs, interactions with other KEAP1 residues are critical for the stereospecific recognition and potency of these ligands. GENERAL SIGNIFICANCE: This work demonstrates that reversible cyanoenone Michael acceptors, such as the tpAIMs and sAIMs, can be specifically tuned to regulate redox sensitive cysteine residues on key signaling molecules, an approach with significant promise for innovative drug development.
Asunto(s)
Antioxidantes/farmacología , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Ácido Oleanólico/química , Relación Estructura-Actividad Cuantitativa , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antioxidantes/química , Sitios de Unión , Células HEK293 , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/química , Ratones , Simulación del Acoplamiento Molecular , Factor 2 Relacionado con NF-E2/química , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Intrinsically disordered proteins play crucial roles in regulatory processes and often function as protein interaction hubs. Here, we present a detailed characterization of a full-length disordered hub protein region involved in multiple dynamic complexes. We performed NMR, CD, and fluorescence binding studies on the nonphosphorylated and highly PKA-phosphorylated human cystic fibrosis transmembrane conductance regulator (CFTR) regulatory region, a â¼200-residue disordered segment involved in phosphorylation-dependent regulation of channel trafficking and gating. Our data provide evidence for dynamic, phosphorylation-dependent, multisite interactions of various segments of the regulatory region for its intra- and intermolecular partners, including the CFTR nucleotide binding domains 1 and 2, a 42-residue peptide from the C terminus of CFTR, the SLC26A3 sulphate transporter and antisigma factor antagonist (STAS) domain, and 14-3-3ß. Because of its large number of binding partners, multivalent binding of individually weak sites facilitates rapid exchange between free and bound states to allow the regulatory region to engage with different partners and generate a graded or rheostat-like response to phosphorylation. Our results enrich the understanding of how disordered binding segments interact with multiple targets. We present structural models consistent with our data that illustrate this dynamic aspect of phospho-regulation of CFTR by the disordered regulatory region.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Modelos Moleculares , Conformación Proteica , Mapas de Interacción de Proteínas/fisiología , Secuencias Reguladoras de Ácidos Nucleicos/fisiología , Proteínas 14-3-3/metabolismo , Biofisica , Antiportadores de Cloruro-Bicarbonato/metabolismo , Dicroismo Circular , Fluorescencia , Humanos , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Unión Proteica , Pliegue de Proteína , Mapas de Interacción de Proteínas/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Transportadores de SulfatoRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Distonía Muscular Deformante/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/genética , Animales , Western Blotting , Línea Celular , Distonía Muscular Deformante/genética , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Glicosilación , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Células HeLa , Proteínas de Choque Térmico/genética , Humanos , Microscopía Fluorescente , Chaperonas Moleculares/genética , Mutación , Estabilidad Proteica , Interferencia de ARN , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The most common cystic fibrosis (CF)-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of Phe508 (DeltaF508) in the first of two nucleotide-binding domains (NBDs). Nucleotide binding and hydrolysis at the NBDs and phosphorylation of the regulatory (R) region are required for gating of CFTR chloride channel activity. We report NMR studies of wild-type and DeltaF508 murine CFTR NBD1 with the C-terminal regulatory extension (RE), which contains residues of the R region. Interactions of the wild-type NBD1 core with the phosphoregulatory regions, the regulatory insertion (RI) and RE, are disrupted upon phosphorylation, exposing a potential binding site for the first coupling helix of the N-terminal intracellular domain (ICD). Phosphorylation of DeltaF508 NBD1 does not as effectively disrupt interactions with the phosphoregulatory regions, which, along with other structural differences, leads to decreased binding of the first coupling helix. These results provide a structural basis by which phosphorylation of CFTR may affect the channel gating of full-length CFTR and expand our understanding of the molecular basis of the DeltaF508 defect.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Animales , Sitios de Unión , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Técnicas In Vitro , Activación del Canal Iónico , Ratones , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de SecuenciaRESUMEN
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.
Asunto(s)
Aminofenoles , Benzodioxoles , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Indoles , Pirazoles , Quinolonas , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , Quinolonas/uso terapéutico , Quinolonas/farmacología , Aminofenoles/uso terapéutico , Aminofenoles/farmacología , Animales , Ratas , Humanos , Benzodioxoles/uso terapéutico , Benzodioxoles/farmacología , Indoles/uso terapéutico , Indoles/farmacología , Pirazoles/farmacología , Pirazoles/uso terapéutico , Piridinas/farmacología , Piridinas/uso terapéutico , Combinación de Medicamentos , Agonistas de los Canales de Cloruro/uso terapéutico , Pirrolidinas/farmacología , Pirrolidinas/uso terapéuticoRESUMEN
Chemical modulation of histone deacetylase (HDAC) activity by HDAC inhibitors (HDACi) is an increasingly important approach for modifying the etiology of human disease. Loss-of-function diseases arise as a consequence of protein misfolding and degradation, which lead to system failures. The DeltaF508 mutation in cystic fibrosis transmembrane conductance regulator (CFTR) results in the absence of the cell surface chloride channel and a loss of airway hydration, leading to the premature lung failure and reduced lifespan responsible for cystic fibrosis. We now show that the HDACi suberoylanilide hydroxamic acid (SAHA) restores surface channel activity in human primary airway epithelia to levels that are 28% of those of wild-type CFTR. Biological silencing of all known class I and II HDACs reveals that HDAC7 plays a central role in restoration of DeltaF508 function. We suggest that the tunable capacity of HDACs can be manipulated by chemical biology to counter the onset of cystic fibrosis and other human misfolding disorders.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , Histona Desacetilasas/metabolismo , Mutación , Animales , Bronquios/metabolismo , Membrana Celular/metabolismo , Cricetinae , Células Epiteliales/metabolismo , Silenciador del Gen , Humanos , Ácidos Hidroxámicos/química , Desnaturalización Proteica , Pliegue de Proteína , ARN Interferente Pequeño/metabolismo , VorinostatRESUMEN
The regulatory (R) region of the cystic fibrosis transmembrane conductance regulator (CFTR) is intrinsically disordered and must be phosphorylated at multiple sites for full CFTR channel activity, with no one specific phosphorylation site required. In addition, nucleotide binding and hydrolysis at the nucleotide-binding domains (NBDs) of CFTR are required for channel gating. We report NMR studies in the absence and presence of NBD1 that provide structural details for the isolated R region and its interaction with NBD1 at residue-level resolution. Several sites in the R region with measured fractional helical propensity mediate interactions with NBD1. Phosphorylation reduces the helicity of many R-region sites and reduces their NBD1 interactions. This evidence for a dynamic complex with NBD1 that transiently engages different sites of the R region suggests a structural explanation for the dependence of CFTR activity on multiple PKA phosphorylation sites.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Sitios de Unión , Humanos , Hidrólisis , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Pliegue de Proteína , Estructura Terciaria de ProteínaRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/metabolismo , Distonía/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Secuencias de Aminoácidos , Animales , Células COS , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Chlorocebus aethiops , Distonía/genética , Proteínas del Choque Térmico HSC70/genética , Proteínas del Choque Térmico HSC70/metabolismo , Células HeLa , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/genética , Mutación , Oxidación-Reducción , Unión Proteica , Conformación ProteicaRESUMEN
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.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Mutación , Fenilalanina/genética , Sitios de Unión/genética , Western Blotting , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Fenilalanina/química , Fenilalanina/metabolismo , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Eliminación de Secuencia , Supresión GenéticaRESUMEN
The pathological hallmark of Parkinson's disease and diffuse Lewy body disease (DLBD) is the aggregation of α-synuclein (α-syn) in the form of Lewy bodies and Lewy neurites. Patients with both Alzheimer's disease (AD) and cortical Lewy pathology represent the Lewy body variant of AD (LBV) and constitute 25% of AD cases. C-terminally truncated forms of α-syn enhance the aggregation of α-syn in vitro. To investigate the presence of C-terminally truncated α-syn in DLBD, AD, and LBV, we generated and validated polyclonal antibodies to truncated α-syn ending at residues 110 (α-syn110) and 119 (α-syn119), two products of 20S proteosome-mediated endoproteolytic cleavage. Double immunofluorescence staining of the cingulate cortex showed that α-syn110 and α-syn140 (full-length) aggregates were not colocalized in LBV. All aggregates containing α-syn140 also contained α-syn119; however, some aggregates contained α-syn119 without α-syn140, suggesting that α-syn119 may stimulate aggregate formation. Immunohistochemistry and image analysis of tissue microarrays of the cingulate cortex from patients with DLBD (n = 27), LBV (n = 27), and AD (n = 19) and age-matched controls (n = 15) revealed that AD is also characterized by frequent abnormal neurites containing α-syn119. Notably, these neurites did not contain α-syn ending at residues 110 or 122-140. The presence of abnormal neurites containing α-syn119 in AD without conventional Lewy pathology suggests that AD and Lewy body disease may be more closely related than previously thought.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Cuerpos de Lewy/patología , Neuritas/metabolismo , Neuritas/patología , alfa-Sinucleína/metabolismo , Anciano , Enfermedad de Alzheimer/patología , Autopsia , Encéfalo/metabolismo , Encéfalo/patología , Estudios de Casos y Controles , Humanos , Inmunohistoquímica , Cuerpos de Lewy/metabolismo , Enfermedad por Cuerpos de Lewy/metabolismo , Enfermedad por Cuerpos de Lewy/patología , Persona de Mediana Edad , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Análisis de Matrices Tisulares , alfa-Sinucleína/químicaRESUMEN
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.
Asunto(s)
Antiportadores , Bicarbonatos/metabolismo , Proteínas Portadoras/metabolismo , Antiportadores de Cloruro-Bicarbonato/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Activación del Canal Iónico/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Proteínas Portadoras/genética , Línea Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Cloruros/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Activación del Canal Iónico/genética , Ligandos , Proteínas de la Membrana/genética , Fosforilación , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Transportadores de Sulfato , Equilibrio Hidroelectrolítico/genéticaRESUMEN
A hallmark of Parkinson disease (PD) is the formation of intracellular protein inclusions called Lewy bodies that also contain mitochondria. alpha-Synuclein (alpha Syn) is a major protein component of Lewy bodies, where it is in an amyloid conformation and a significant fraction is truncated by poorly understood proteolytic events. Previously, we demonstrated that the 20S proteasome cleaves alpha Syn in vitro to produce fragments like those observed in Lewy bodies and that the fragments accelerate the formation of amyloid fibrils from full-length alpha Syn. Three point mutations in alpha Syn are associated with early-onset familial PD: A30P, E46K, and A53T. However, these mutations have very different effects on the amyloidogenicity and vesicle-binding activity of alpha Syn, suggesting neither of these processes directly correlate with neurodegeneration. Here, we evaluate the effect of the disease-associated mutations on the fragmentation, conformation, and association reactions of alpha Syn in the presence of the 20S proteasome and liposomes. The 20S proteasome produced the C-terminal fragments from both the mutant and wildtype alpha Syn. These truncations accelerated fibrillization of all alpha-synucleins, but again there was no clear correlation between the PD-associated mutations and amyloid formation in the presence of liposomes. Recent data suggests that cellular toxicity is caused by a soluble oligomeric species, which is a precursor to the amyloid form and is immunologically distinguishable from both soluble monomeric and amyloid forms of alpha Syn. Notably, the rate of formation of the soluble, presumptively cytotoxic oligomers correlated with the disease-associated mutations when both 20S proteasome and liposomes were present. Under these conditions, the wildtype protein was also cleaved and formed the oligomeric structures, albeit at a slower rate, suggesting that 20S-mediated truncation of alpha Syn may play a role in sporadic PD as well. Evaluation of the biochemical reactions of the PD-associated alpha-synuclein mutants in our in vitro system provides insight into the possible pathogenetic mechanism of both familial and sporadic PD.
Asunto(s)
Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Mutación Puntual , Complejo de la Endopetidasa Proteasomal/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Amiloide/biosíntesis , Animales , Bovinos , Humanos , Técnicas In Vitro , Cinética , Cuerpos de Lewy/genética , Cuerpos de Lewy/metabolismo , Liposomas , Modelos Neurológicos , Enfermedad de Parkinson/etiología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Multimerización de Proteína , alfa-Sinucleína/químicaRESUMEN
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fenilalanina/metabolismo , Pliegue de Proteína , Animales , Sitios de Unión , Línea Celular , Cristalografía por Rayos X , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Modelos Moleculares , Mutación/genética , Fenilalanina/genética , Desnaturalización Proteica , Estructura Terciaria de Proteína , Temperatura , TermodinámicaRESUMEN
Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, ~tenfold higher than hit rates obtained in corresponding high-throughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of our knowledge this is the first report of structure-based discovery of CFTR modulators.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/efectos de los fármacos , Transporte Iónico/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Animales , Sitios de Unión/genética , Línea Celular , Células Cultivadas , Simulación por Computador , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Fibrosis Quística/fisiopatología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Ratas Endogámicas F344 , Mucosa Respiratoria/efectos de los fármacos , Eliminación de Secuencia , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) Cl(-) channel. F508del, the most frequent CF-causing mutation, disrupts both the processing and function of CFTR. Recently, the crystal structure of the first nucleotide-binding domain of CFTR bearing F508del (F508del-NBD1) was elucidated. Although F508del-NBD1 shows only minor conformational changes relative to that of wild-type NBD1, additional mutations (F494N/Q637R or F429S/F494N/Q637R) were required for domain solubility and crystallization. Here we show that these solubilizing mutations in cis with F508del partially rescue the trafficking defect of full-length F508del-CFTR and attenuate its gating defect. We interpret these data to suggest that the solubilizing mutations utilized to facilitate F508del-NBD1 production also assist folding of full-length F508del-CFTR protein. Thus, the available crystal structure of F508del-NBD1 might correspond to a partially corrected conformation of this domain.