RESUMO
Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, leading to defective apical chloride transport. Patients also experience overactivation of inflammatory processes, including increased calcium signaling. Many investigations have described indirect effects of calcium signaling on CFTR or other calcium-activated chloride channels; here, we investigate the direct response of CFTR to calmodulin-mediated calcium signaling. We characterize an interaction between the regulatory region of CFTR and calmodulin, the major calcium signaling molecule, and report protein kinase A (PKA)-independent CFTR activation by calmodulin. We describe the competition between calmodulin binding and PKA phosphorylation and the differential effects of this competition for wild-type CFTR and the major F508del mutant, hinting at potential therapeutic strategies. Evidence of CFTR binding to isolated calmodulin domains/lobes suggests a mechanism for the role of CFTR as a molecular hub. Together, these data provide insights into how loss of active CFTR at the membrane can have additional consequences besides impaired chloride transport.
Assuntos
Cálcio/metabolismo , AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulação da Expressão Gênica , Transdução de Sinais , Sítios de Ligação , Sinalização do Cálcio , Calmodulina/metabolismo , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Humanos , Espectroscopia de Ressonância Magnética , Potenciais da Membrana , Modelos Biológicos , Modelos Moleculares , Conformação Molecular , Mutação , Fosforilação , Ligação Proteica , Transporte Proteico , Elementos de RespostaRESUMO
SLC6A14-mediated l-arginine transport has been shown to augment the residual anion channel activity of the major mutant, F508del-CFTR, in the murine gastrointestinal tract. It is not yet known if this transporter augments residual and pharmacological corrected F508del-CFTR in primary airway epithelia. We sought to determine the role of l-arginine uptake via SLC6A14 in modifying F508del-CFTR channel activity in airway cells from patients with cystic fibrosis (CF). Human bronchial epithelial (HBE) cells from lung explants of patients without CF (HBE) and those with CF (CF-HBE) were used for H3-flux, airway surface liquid, and Ussing chamber studies. We used α-methyltryptophan as a specific inhibitor for SLC6A14. CFBE41o-, a commonly used CF airway cell line, was employed for studying the mechanism of the functional interaction between SLC6A14 and F508del-CFTR. SLC6A14 is functionally expressed in CF-HBE cells. l-arginine uptake via SLC6A14 augmented F508del-CFTR function at baseline and after treatment with lumacaftor. SLC6A14-mediated l-arginine uptake also increased the airway surface liquid in CF-HBE cells. Using CFBE41o cells, we showed that the positive SLC6A14 effect was mainly dependent on the nitric oxide (NO) synthase activity, nitrogen oxides, including NO, and phosphorylation by protein kinase G. These finding were confirmed in CF-HBE, as inducible NO synthase inhibition abrogated the functional interaction between SLC6A14 and pharmacological corrected F508del-CFTR. In summary, SLC6A14-mediated l-arginine transport augments residual F508del-CFTR channel function via a noncanonical, NO pathway. This effect is enhanced with increasing pharmacological rescue of F508del-CFTR to the membrane. The current study demonstrates how endogenous pathways can be used for the development of companion therapy in CF.
Assuntos
Sistemas de Transporte de Aminoácidos/fisiologia , Arginina/metabolismo , Brônquios/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Fibrose Cística/terapia , Sistemas de Transporte de Aminoácidos/antagonistas & inibidores , Sistemas de Transporte de Aminoácidos/genética , Transporte Biológico , Brônquios/citologia , Células Cultivadas , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Genes Reporter , Humanos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Recombinantes/metabolismo , Propriedades de Superfície , Transdução Genética , Triptofano/análogos & derivados , Triptofano/farmacologiaRESUMO
Aggregation can be a major challenge in the development of antibody-based pharmaceuticals as it can compromise the quality of the product during bioprocessing, formulation, and drug administration. To avoid aggregation, developability assessment is often run in parallel with functional optimization in the early screening phases to flag and deselect problematic molecules. As developability assessment can be demanding with regard to time and resources, there is a high focus on the development of molecule design strategies for engineering molecules with a high developability potential. Previously, Dudgeon et al. [(2012) Proc. Natl. Acad. Sci. U. S. A. 109, 10879-10884] demonstrated how Asp substitutions at specific positions in human variable domains and single-chain variable fragments could decrease the aggregation propensity. Here, we have investigated whether these Asp substitutions would improve the developability potential of a murine antigen binding fragment (Fab). A full combinatorial library consisting of 393 Fab variants with single, double, and triple Asp substitutions was first screened in silico with Rosetta; thereafter, 26 variants with the highest predicted thermodynamic stability were selected for production. All variants were subjected to a set of developability studies. Interestingly, most variants had thermodynamic stability on par with or improved relative to that of the wild type. Twenty-five of the variants exhibited improved nonspecificity. Half of the variants exhibited improved aggregation resistance. Strikingly, while we observed remarkable improvement in the developability potential, the Asp substitutions had no substantial effect on the antigenic binding affinity. Altogether, by combining the insertion of negative charges and the in silico screen based on computational models, we were able to improve the developability of the Fab rapidly.
Assuntos
Ácido Aspártico/química , Fragmentos Fab das Imunoglobulinas/química , Substituição de Aminoácidos , Animais , Antígenos/imunologia , Simulação por Computador , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Camundongos , Biblioteca de Peptídeos , Multimerização Proteica/genética , Estabilidade ProteicaRESUMO
Eukaryotic CLC anion channels and transporters are homodimeric proteins composed of multiple α-helical membrane domains and large cytoplasmic C-termini containing two cystathionine-ß-synthase domains (CBS1 and CBS2) that dimerize to form a Bateman domain. The Bateman domains of adjacent CLC subunits interact to form a Bateman domain dimer. The functions of CLC CBS and Bateman domains are poorly understood. We utilized the Caenorhabditis elegans CLC-1/2/Ka/Kb anion channel homolog CLH-3b to characterize the regulatory roles of CLC cytoplasmic domains. CLH-3b activity is reduced by phosphorylation or deletion of a 14-amino-acid activation domain (AD) located on the linker connecting CBS1 and CBS2. We demonstrate here that phosphorylation-dependent reductions in channel activity require an intact Bateman domain dimer and concomitant phosphorylation or deletion of both ADs. Regulation of a CLH-3b AD deletion mutant is reconstituted by intracellular perfusion with recombinant 14-amino-acid AD peptides. The sulfhydryl reactive reagent 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET) alters in a phosphorylation-dependent manner the activity of channels containing single cysteine residues that are engineered into the short intracellular loop connecting membrane α-helices H and I (H-I loop), the AD, CBS1, and CBS2. In contrast, MTSET has no effect on channels in which cysteine residues are engineered into intracellular regions that are dispensable for regulation. These studies together with our previous work suggest that binding and unbinding of the AD to the Bateman domain dimer induces conformational changes that are transduced to channel membrane domains via the H-I loop. Our findings provide new, to our knowledge, insights into the roles of CLC Bateman domains and the structure-function relationships that govern the regulation of CLC protein activity by diverse ligands and signaling pathways.
Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Canais de Cloreto/química , Canais de Cloreto/metabolismo , Cistationina beta-Sintase/química , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Células HEK293 , Humanos , Ativação do Canal Iônico , Ligantes , Modelos Moleculares , Fosforilação , Domínios Proteicos , Transdução de SinaisRESUMO
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.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Modelos Moleculares , Conformação Proteica , Mapas de Interação de Proteínas/fisiologia , Sequências Reguladoras de Ácido Nucleico/fisiologia , Proteínas 14-3-3/metabolismo , Biofísica , Antiportadores de Cloreto-Bicarbonato/metabolismo , Dicroísmo Circular , Fluorescência , Humanos , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Ligação Proteica , Dobramento de Proteína , Mapas de Interação de Proteínas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Transportadores de SulfatoRESUMO
Phosphorylation adjacent to nuclear localization signals (NLSs) is involved in the regulation of nucleocytoplasmic transport. The nuclear isoform of human dUTPase, an enzyme that is essential for genomic integrity, has been shown to be phosphorylated on a serine residue (Ser11) in the vicinity of its nuclear localization signal; however, the effect of this phosphorylation is not yet known. To investigate this issue, an integrated set of structural, molecular and cell biological methods were employed. It is shown that NLS-adjacent phosphorylation of dUTPase occurs during the M phase of the cell cycle. Comparison of the cellular distribution of wild-type dUTPase with those of hyperphosphorylation- and hypophosphorylation-mimicking mutants suggests that phosphorylation at Ser11 leads to the exclusion of dUTPase from the nucleus. Isothermal titration microcalorimetry and additional independent biophysical techniques show that the interaction between dUTPase and importin-α, the karyopherin molecule responsible for `classical' NLS binding, is weakened significantly in the case of the S11E hyperphosphorylation-mimicking mutant. The structures of the importin-α-wild-type and the importin-α-hyperphosphorylation-mimicking dUTPase NLS complexes provide structural insights into the molecular details of this regulation. The data indicate that the post-translational modification of dUTPase during the cell cycle may modulate the nuclear availability of this enzyme.
Assuntos
Pirofosfatases/metabolismo , alfa Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Ciclo Celular , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Sinais de Localização Nuclear , Fosforilação , Pirofosfatases/química , alfa Carioferinas/químicaRESUMO
It has been suggested that in vitro studies of the rescue effect of CFTR modulator drugs in nasal epithelial cultures derived from people with cystic fibrosis have the potential to predict clinical responses to the same drugs. Hence, there is an interest in evaluating different methods for measuring in vitro modulator responses in patient-derived nasal cultures. Commonly, the functional response to CFTR modulator combinations in these cultures is assessed by bioelectric measurements, using the Ussing chamber. While this method is highly informative, it is time-consuming. A fluorescence-based, multi-transwell method for assaying regulated apical chloride conductance (Fl-ACC) promises to provide a complementary approach to theratyping in patient-derived nasal cultures. In the present work, we compared Ussing chamber measurements and fluorescence-based measurements of CFTR-mediated apical conductance in matching, fully differentiated nasal cultures derived from CF patients, homozygous for F508del (n = 31) or W1282X (n = 3), or heterozygous for Class III mutations G551D or G178R (n = 5). These cultures were obtained through a bioresource called the Cystic Fibrosis Canada-Sick Kids Program in Individual CF Therapy (CFIT). We found that the Fl-ACC method was effective in detecting positive responses to interventions for all genotypes. There was a correlation between patient-specific drug responses measured in cultures harbouring F508del, as measured using the Ussing chamber technique and the fluorescence-based assay (Fl-ACC). Finally, the fluorescence-based assay has the potential for greater sensitivity for detecting responses to pharmacological rescue strategies targeting W1282X.
Assuntos
Fibrose Cística , Humanos , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fluorescência , Mutação , GenótipoRESUMO
Induced Pluripotent Stem Cells (iPSCs) can be differentiated into epithelial organoids that recapitulate the relevant context for CFTR and enable testing of therapies targeting Cystic Fibrosis (CF)-causing mutant proteins. However, to date, CF-iPSC-derived organoids have only been used to study pharmacological modulation of mutant CFTR channel activity and not the activity of other disease-relevant membrane protein constituents. In the current work, we describe a high-throughput, fluorescence-based assay of CFTR channel activity in iPSC-derived intestinal organoids and describe how this method can be adapted to study other apical membrane proteins. Specifically, we show how this assay can be employed to study CFTR and ENaC channels and an electrogenic acid transporter in the same iPSC-derived intestinal tissue. This phenotypic platform promises to expand CF therapy discovery to include strategies that target multiple determinants of epithelial fluid transport.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Intestinos/metabolismo , Organoides/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Animais , Diferenciação Celular , Cães , Canais Epiteliais de Sódio/metabolismo , Edição de Genes , Humanos , Células Madin Darby de Rim CaninoRESUMO
For those people with cystic fibrosis carrying rare CFTR mutations not responding to currently available therapies, there is an unmet need for relevant tissue models for therapy development. Here, we describe a new testing platform that employs patient-specific induced pluripotent stem cells (iPSCs) differentiated to lung progenitor cells that can be studied using a dynamic, high-throughput fluorescence-based assay of CFTR channel activity. Our proof-of-concept studies support the potential use of this platform, together with a Canadian bioresource that contains iPSC lines and matched nasal cultures from people with rare mutations, to advance patient-oriented therapy development. Interventions identified in the high-throughput, stem cell-based model and validated in primary nasal cultures from the same person have the potential to be advanced as therapies.
Assuntos
Diferenciação Celular/genética , Fibrose Cística/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Pulmão/metabolismo , Células-Tronco/metabolismo , Células Cultivadas , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Pulmão/citologia , Mutação , RNA-Seq/métodos , Células-Tronco/citologiaRESUMO
We present the Canadian Distributed Infrastructure for Genomics (CanDIG) platform, which enables federated querying and analysis of human genomics and linked biomedical data. CanDIG leverages the standards and frameworks of the Global Alliance for Genomics and Health (GA4GH) and currently hosts data for five pan-Canadian projects. We describe CanDIG's key design decisions and features as a guide for other federated data systems.
RESUMO
Advanced and metastatic tumors with complex treatment histories drive cancer mortality. Here we describe the POG570 cohort, a comprehensive whole-genome, transcriptome and clinical dataset, amenable for exploration of the impacts of therapies on genomic landscapes. Previous exposure to DNA-damaging chemotherapies and mutations affecting DNA repair genes, including POLQ and genes encoding Polζ, were associated with genome-wide, therapy-induced mutagenesis. Exposure to platinum therapies coincided with signatures SBS31 and DSB5 and, when combined with DNA synthesis inhibitors, signature SBS17b. Alterations in ESR1, EGFR, CTNNB1, FGFR1, VEGFA and DPYD were consistent with drug resistance and sensitivity. Recurrent noncoding events were found in regulatory region hotspots of genes including TERT, PLEKHS1, AP2A1 and ADGRG6. Mutation burden and immune signatures corresponded with overall survival and response to immunotherapy. Our data offer a rich resource for investigation of advanced cancers and interpretation of whole-genome and transcriptome sequencing in the context of a cancer clinic.
Assuntos
Neoplasias , Humanos , Neoplasias/tratamento farmacológicoRESUMO
Calpains are cysteine proteases involved in a number of physiological and pathological processes, yet our knowledge of substrates cleaved in vivo, in intact cells, is scarce. In this work we made an attempt to develop a technique for finding calpain substrates in intact Drosophila Schneider S2 cells. The procedure consists in comparative 2D gelelectrophoresis: three identical samples were treated in different ways: A (control, no addition), B, activated (Ca(2+) and ionomycin added), C, inactivated (additions as in B+specific calpain inhibitor). 2D gel pattern were analyzed by densitometry. Spots showing density relation A>B<Assuntos
Calpaína/metabolismo
, Proteínas de Drosophila/metabolismo
, Drosophila/metabolismo
, Animais
, Cálcio/farmacologia
, Calpaína/antagonistas & inibidores
, Calpaína/genética
, Linhagem Celular
, Primers do DNA
, Proteínas de Drosophila/genética
, Ionomicina/farmacologia
, Espectrometria de Massas
, Reação em Cadeia da Polimerase
, Proteínas Recombinantes/metabolismo
, Especificidade por Substrato
RESUMO
PURPOSE: Current literature suggests that longer duration of EEG recording increases the yield of detecting interictal epileptiform discharges. However, optimal duration for a repeat study in patients with initially normal 30-minute EEG is not clear. Thus, the purpose of this study is to determine whether a 2-hour EEG has a diagnostic advantage over a routine 30-minute EEG in detecting epileptiform abnormalities in patients who had a first normal 30-minute EEG. METHODS: This is a single-center, retrospective study done at UT Southwestern Medical Center at Dallas and Parkland Memorial Hospital. The data from 1997 to 2015 were extracted from the existing EEG report database for patients who had a first normal 30-minute EEG recording. EEG was interpreted by board-certified clinical neurophysiologists, who classified each EEG as normal or abnormal, with relevant subsequent subclassification. RESULTS: Over 18 years, a total of 12,425 individual 30-minute EEGs were performed. Of these, 1,023 patients had at least one repeated EEG after the first normal EEG. Among these patients, 763 had a 30-minute EEG as the second study and 260 had a 2-hour EEG as the second study. The yield of epileptiform discharges was 3.3% in the 30-minute EEG group and 4.2% in the 2-hour EEG group (P = 0.5) in the repeating studies. CONCLUSIONS: Two-hour EEG has a similar yield as 30-minute EEG to detect epileptiform discharges in patients with a normal 30-minute EEG.
Assuntos
Eletroencefalografia/métodos , Epilepsia/diagnóstico , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de TempoRESUMO
The activity of calpain is controlled by the free intracellular calcium level and by the protein's intrinsically disordered endogenous inhibitor, calpastatin, mediated by short conserved segments: subdomains A-C. The exact binding mode of calpastatin to the enzyme has until now been unclear. Our NMR data of the 141 amino acid long inhibitor, with and without calcium and calpain, have revealed structural changes and a tripartite binding mode, in which the disordered inhibitor wraps around, and contacts, the enzyme at three points, facilitated by flexible linkers. This unprecedented binding mode permits a unique combination of specificity, speed and binding strength in regulation.
Assuntos
Proteínas de Ligação ao Cálcio/química , Cálcio/química , Calpaína/antagonistas & inibidores , Calpaína/química , Humanos , Ressonância Magnética Nuclear Biomolecular , Conformação ProteicaRESUMO
Although membrane proteins represent most therapeutically relevant drug targets, the availability of atomic resolution structures for this class of proteins has been limited. Structural characterization has been hampered by the biophysical nature of these polytopic transporters, receptors, and channels, and recent innovations to in vitro techniques aim to mitigate these challenges. One such class of membrane proteins, the ATP-binding cassette (ABC) superfamily, are broadly expressed throughout the human body, required for normal physiology and disease-causing when mutated, yet lacks sufficient structural representation in the Protein Data Bank. However, recent improvements to biophysical techniques (e.g., cryo-electron microscopy) have allowed for previously "hard-to-study" ABC proteins to be characterized at high resolution, providing insight into molecular mechanisms-of-action as well as revealing novel druggable sites for therapy design. These new advances provide ample opportunity for computational methods (e.g., virtual screening, molecular dynamics simulations, and structure-based drug design) to catalyze the discovery of novel small molecule therapeutics that can be easily translated from computer to bench and subsequently to the patient's bedside. In this review, we explore the utility of recent advances in biophysical methods coupled with well-established in silico techniques towards drug development for diseases caused by dysfunctional ABC proteins.
RESUMO
Pulmonary disease is the major cause of morbidity and mortality in patients with cystic fibrosis, a disease caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Heterogeneity in CFTR genotype-phenotype relationships in affected individuals plus the escalation of drug discovery targeting specific mutations highlights the need to develop robust in vitro platforms with which to stratify therapeutic options using relevant tissue. Toward this goal, we adapted a fluorescence plate reader assay of apical CFTR-mediated chloride conductance to enable profiling of a panel of modulators on primary nasal epithelial cultures derived from patients bearing different CFTR mutations. This platform faithfully recapitulated patient-specific responses previously observed in the "gold-standard" but relatively low-throughput Ussing chamber. Moreover, using this approach, we identified a novel strategy with which to augment the response to an approved drug in specific patients. In proof of concept studies, we also validated the use of this platform in measuring drug responses in lung cultures differentiated from cystic fibrosis iPS cells. Taken together, we show that this medium throughput assay of CFTR activity has the potential to stratify cystic fibrosis patient-specific responses to approved drugs and investigational compounds in vitro in primary and iPS cell-derived airway cultures.
RESUMO
Typical calpains in mammals become activated on binding of 8-12 Ca2+ ions per enzyme molecule, giving an example of integrated, manifold regulation by calcium. Besides two identified Ca2+ sites in catalytic domain II and several EF-hand motifs in domains IV and VI, an acidic loop in the centrally positioned domain III seems to harbour Ca2+. The mediator of distant Ca2+-induced structural transitions is an elongated structural element, the 'transducer'. By site-directed mutagenesis along the transducer, we have generated various forms of rat m-calpain in which critical intramolecular interactions, as judged from the X-ray structure, would be abolished or modified. The kinetic parameters of these mutant enzymes support a model featuring shrinkage of transducer as a contributor to structural changes involved in calpain activation.
Assuntos
Cálcio/farmacologia , Calpaína/química , Calpaína/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Sinalização do Cálcio/fisiologia , Calpaína/genética , Domínio Catalítico , Ativação Enzimática/efeitos dos fármacos , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , RatosRESUMO
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel in the apical surface of epithelial cells in the airway and gastrointestinal tract, and mutation of CFTR is the underlying cause of cystic fibrosis. However, the precise molecular details of the structure and function of CFTR in native and disease states remains elusive and cystic fibrosis researchers are hindered by a lack of high specificity, high affinity binding reagents for use in structural and biological studies. Here, we describe a panel of synthetic antigen-binding fragments (Fabs) isolated from a phage-displayed library that are specific for intracellular domains of CFTR that include the nucleotide-binding domains (NBD1 and NBD2), the R-region, and the regulatory insertion loop of NBD1. Binding assays performed under conditions that promote the native fold of the protein demonstrated that all Fabs recognized full-length CFTR. However, only the NBD1-specific Fab recognized denatured CFTR by western blot, suggesting a conformational epitope requirement for the other Fabs. Surface plasmon resonance experiments showed that the R-region Fab binds with high affinity to both the phosphorylated and unphosphorylated R-region. In addition, NMR analysis of bound versus unbound R-region revealed a distinct conformational effect upon Fab binding. We further defined residues involved with antibody recognition using an overlapping peptide array. In summary, we describe methodology complementary to previous hybridoma-based efforts to develop antibody reagents to CFTR, and introduce a synthetic antibody panel to aid structural and biological studies.
Assuntos
Anticorpos/química , Regulador de Condutância Transmembrana em Fibrose Cística/imunologia , Fragmentos Fab das Imunoglobulinas/química , Anticorpos/genética , Afinidade de Anticorpos , Epitopos/química , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Espectroscopia de Ressonância Magnética , Biblioteca de Peptídeos , Fosforilação , Domínios Proteicos , Engenharia de Proteínas , Dobramento de Proteína , Ressonância de Plasmônio de SuperfícieRESUMO
Phosphorylation by the cyclin-dependent kinase 1 (Cdk1) adjacent to nuclear localization signals (NLSs) is an important mechanism of regulation of nucleocytoplasmic transport. However, no systematic survey has yet been performed in human cells to analyze this regulatory process, and the corresponding cell-cycle dynamics have not yet been investigated. Here, we focused on the human proteome and found that numerous proteins, previously not identified in this context, are associated with Cdk1-dependent phosphorylation sites adjacent to their NLSs. Interestingly, these proteins are involved in key regulatory events of DNA repair, epigenetics, or RNA editing and splicing. This finding indicates that cell-cycle dependent events of genome editing and gene expression profiling may be controlled by nucleocytoplasmic trafficking. For in-depth investigations, we selected a number of these proteins and analyzed how point mutations, expected to modify the phosphorylation ability of the NLS segments, perturb nucleocytoplasmic localization. In each case, we found that mutations mimicking hyper-phosphorylation abolish nuclear import processes. To understand the mechanism underlying these phenomena, we performed a video microscopy-based kinetic analysis to obtain information on cell-cycle dynamics on a model protein, dUTPase. We show that the NLS-adjacent phosphorylation by Cdk1 of human dUTPase, an enzyme essential for genomic integrity, results in dynamic cell cycle-dependent distribution of the protein. Non-phosphorylatable mutants have drastically altered protein re-import characteristics into the nucleus during the G1 phase. Our results suggest a dynamic Cdk1-driven mechanism of regulation of the nuclear proteome composition during the cell cycle.
Assuntos
Quinases Ciclina-Dependentes/genética , Reparo do DNA/genética , Sinais de Localização Nuclear/genética , Proteoma , Sequência de Aminoácidos , Proteína Quinase CDC2 , Ciclo Celular/genética , Divisão Celular , Quinases Ciclina-Dependentes/metabolismo , Humanos , FosforilaçãoRESUMO
Chloride channel gating and trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) are regulated by phosphorylation. Intrinsically disordered segments of the protein are responsible for phospho-regulation, particularly the regulatory (R) region that is a target for several kinases and phosphatases. The R region remains disordered following phosphorylation, with different phosphorylation states sampling various conformations. Recent studies have demonstrated the crucial role that intramolecular and intermolecular interactions of the R region play in CFTR regulation. Different partners compete for the same binding segment, with the R region containing multiple overlapping binding elements. The non-phosphorylated R region interacts with the nucleotide binding domains and inhibits channel activity by blocking heterodimerization. Phosphorylation shifts the equilibrium such that the R region is excluded from the dimer interface, facilitating gating and processing by stimulating R region interactions with other domains and proteins. The dynamic conformational sampling and transient binding of the R region to multiple partners enables complex control of CFTR channel activity and trafficking.