RESUMEN
Prior investigations show that signaling activation through pattern recognition receptors can directly impact a number of inflammatory lung diseases. While toll-like receptor (TLR) 7 agonists have raised interest for their ability to inhibit allergen-induced pathological changes in experimental asthma conditions, the putative benefit of this treatment is limited by adverse effects. Our aim was to evaluate the therapeutic potential of two PEGylated purine-like compounds, TMX-302 and TMX-306, characterized by TLR7 partial agonistic activity; therefore, the compounds are expected to induce lower local and systemic adverse reactions. In vitro approaches and translation to murine models of obstructive and restrictive lung diseases were explored. In vitro studies with human PBMCs showed that both TMX-302 and TMX-306 marginally affects cytokine production as compared with equivalent concentrations of the TLR7 full agonist, TMX-202. The PEGylated compounds did not induce monocyte-derived DC maturation or B cell proliferation, differently from what observed after stimulation with TMX-202. Impact of PEGylated ligands on lung function and inflammatory changes was studied in animal models of acute lung injury, asthma, and silicosis following Lipopolysaccharide (LPS), allergen (ovalbumin), and silica inhalation, respectively. Subcutaneous injection of TMX-302 prevented LPS- and allergen-induced airway hyper-reactivity (AHR), leukocyte infiltration, and production of pro-inflammatory cytokines in the lung. However, intranasal instillation of TMX-302 led to neutrophil infiltration and failed to prevent allergen-induced AHR, despite inhibiting leukocyte counts in the BAL. Aerosolized TMX-306 given prophylactically, but not therapeutically, inhibited pivotal asthma features. Interventional treatment with intranasal instillation of TMX-306 significantly reduced the pulmonary fibrogranulomatous response and the number of silica particles in lung interstitial space in silicotic mice. These findings highlight the potential of TMX-306, emphasizing its value in drug development for lung diseases, and particularly silicosis.
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OBJECTIVES: Monocytes are one of the major phagocytic cells that patrol for invading pathogens, and upon activation, differentiate into macrophages or antigen-presenting dendritic cells (DCs) capable of migrating to lymph nodes eliciting an adaptive immune response. The key role in regulating adaptive immune responses has drawn attention to modulate monocyte responses therapeutically within cancer, inflammation and infectious diseases. We present a technology for targeting of monocytes and delivery of a toll-like receptor (TLR) agonist in fresh blood using liposomes with a positively charged surface chemistry. METHODS: Liposomes were extruded at 100 nm, incubated with fresh blood, followed by leukocyte analyses by FACS. Liposomes with and without the TLR7 agonist TMX-202 were incubated with fresh blood, and monocyte activation measured by cytokine secretion by ELISA and CD14 and DC-SIGN expression. RESULTS: The liposomes target monocytes specifically over lymphocytes and granulocytes in human whole blood, and show association with 75 - 95% of the monocytes after 1 h incubation. Formulations of TMX-202 in cationic liposomes were potent in targeting and activation of monocytes, with strong induction of IL-6 and IL-12p40, and differentiation into CD14(+) and DC-SIGN+ DCs. CONCLUSION: Our present liposomes selectively target monocytes in fresh blood, enabling delivery of TLR7 agonists to the intracellular TLR7 receptor, with subsequent monocyte activation and boost in secretion of proinflammatory cytokines. We envision this technology as a promising tool in future cancer immunotherapy.
Asunto(s)
Adenina/análogos & derivados , Células Dendríticas/inmunología , Glicerofosfolípidos/farmacología , Monocitos/efectos de los fármacos , Receptor Toll-Like 7/agonistas , Adenina/administración & dosificación , Adenina/farmacología , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular/efectos de los fármacos , Citocinas/metabolismo , Glicerofosfolípidos/administración & dosificación , Humanos , Subunidad p40 de la Interleucina-12/metabolismo , Lectinas Tipo C/metabolismo , Liposomas , Macrófagos/metabolismo , Masculino , Monocitos/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
BACKGROUND: The Toll-like receptor 7 (TLR7) activator imiquimod (IMQ) is safe and effective in treating actinic keratosis; however, an intermittent treatment regimen is necessary because of excessive local reactions. OBJECTIVES: To evaluate in vitro potency, pharmacodynamics/pharmacokinetics, toxicity and efficacy in vivo of the newly developed TLR7 ligand-phospholipid conjugate, TMX-202, in a gel formulation. MATERIAL AND METHODS: The effects of TMX-202 were assessed both in vitro on a murine macrophage cell line and in primary bone marrow-derived dendritic cells and in vivo on mice (C57BL/6-wild type, Myd88(-/-) and Tlr7(-/-)). RESULTS: TMX-202 was more potent than IMQ in vitro using murine and human cells. In contrast, in vivo it showed less systemic pro-inflammatory activity and better safety than IMQ. Moreover, the TMX-202 gel formulation exhibited at least comparable efficacy to Aldara in a mouse model for skin proliferative diseases. CONCLUSION: TMX-202 is safe and efficacious without causing excessive adverse effects, suggesting that it may be an alternative to Aldara for the treatment of proliferative skin conditions.
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Adenina/análogos & derivados , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Glicerofosfolípidos/farmacología , Glicerofosfolípidos/uso terapéutico , Queratosis Actínica/tratamiento farmacológico , Glicoproteínas de Membrana/genética , Receptor Toll-Like 7/genética , Adenina/sangre , Adenina/farmacología , Adenina/uso terapéutico , Aminoquinolinas/sangre , Aminoquinolinas/farmacología , Animales , Antineoplásicos/sangre , Línea Celular , Factores Quimiotácticos/sangre , Células Dendríticas/fisiología , Geles/farmacología , Geles/uso terapéutico , Glicerofosfolípidos/sangre , Humanos , Imiquimod , Interferón gamma/metabolismo , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Queratinocitos/fisiología , Queratosis Actínica/genética , Leucocitos Mononucleares/efectos de los fármacos , Macrófagos/fisiología , Dosis Máxima Tolerada , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Factor 88 de Diferenciación Mieloide/genética , Proteínas Proto-Oncogénicas c-myc/genética , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In development, tissue regeneration or certain diseases, angiogenic growth leads to the expansion of blood vessels and the lymphatic vasculature. This involves endothelial cell proliferation as well as angiogenic sprouting, in which a subset of cells, termed tip cells, acquires motile, invasive behaviour and extends filopodial protrusions. Although it is already appreciated that angiogenesis is triggered by tissue-derived signals, such as vascular endothelial growth factor (VEGF) family growth factors, the resulting signalling processes in endothelial cells are only partly understood. Here we show with genetic experiments in mouse and zebrafish that ephrin-B2, a transmembrane ligand for Eph receptor tyrosine kinases, promotes sprouting behaviour and motility in the angiogenic endothelium. We link this pro-angiogenic function to a crucial role of ephrin-B2 in the VEGF signalling pathway, which we have studied in detail for VEGFR3, the receptor for VEGF-C. In the absence of ephrin-B2, the internalization of VEGFR3 in cultured cells and mutant mice is defective, which compromises downstream signal transduction by the small GTPase Rac1, Akt and the mitogen-activated protein kinase Erk. Our results show that full VEGFR3 signalling is coupled to receptor internalization. Ephrin-B2 is a key regulator of this process and thereby controls angiogenic and lymphangiogenic growth.
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Efrina-B2/metabolismo , Linfangiogénesis , Neovascularización Fisiológica , Factor C de Crecimiento Endotelial Vascular/metabolismo , Animales , Células Cultivadas , Pérdida del Embrión , Embrión de Mamíferos/irrigación sanguínea , Embrión de Mamíferos/metabolismo , Endocitosis , Células Endoteliales/citología , Células Endoteliales/metabolismo , Efrina-B2/deficiencia , Efrina-B2/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Linfangiogénesis/genética , Vasos Linfáticos , Ratones , Ratones Transgénicos , Neovascularización Fisiológica/genética , Neuropéptidos/metabolismo , Embarazo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor EphB4/deficiencia , Receptor EphB4/genética , Receptor EphB4/metabolismo , Transducción de Señal , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Pez Cebra , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1RESUMEN
OBJECTIVE: To study the immune response caused by the intravesical administration of the immunomodulator R-837 in various formulations and to estimate its therapeutic potential for bladder cancer. METHODS: Female C57BL/6 mice were intravesically treated with different formulations of R-837, a Toll-like receptor 7 agonist used for treating genital warts and skin malignancy. The tested formulation mixtures contained different ratios of lactic acid, a thermosensitive poloxamer polymer (Lutrol F127) and 2-(hydroxypropyl)-beta-cyclodextrin (HPbetaCD). Induction of tumor necrosis factor alpha (TNFalpha) and keratinocyte-derived chemokine (KC) was analyzed by Luminex microbeads assay. The therapeutic potential of intravesical administration of R-837 was assessed in an orthotopic, syngeneic mouse model of bladder cancer using MB49 cells. RESULTS: Intravesical administration of R-837 in lactic acid alone induced systemic and bladder TNFalpha and KC in a dose-dependent manner. Formulations including poloxamer decreased systemic absorption of R-837 and significantly reduced systemic and local induction of KC. Addition of HPbetaCD in the poloxamer formulation particularly reversed levels of systemic and local levels of TNFalpha and KC. Histological examination showed that poloxamer-HPbetaCD formulation allowed infiltration of mononuclear cells into urothelium and lamina propria. In studies using orthotopic mouse bladder cancer, the tumor loads in R-837-treated mice were significantly lower than those in vehicle-treated or non-treated mice. CONCLUSION: The optimized poloxamer-HPbetaCD formulation of R-837 shows therapeutic potential for bladder cancer while avoiding adverse side-effects.
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Adyuvantes Inmunológicos/farmacología , Aminoquinolinas/farmacología , Química Farmacéutica/métodos , Glicoproteínas de Membrana/agonistas , Receptor Toll-Like 7/agonistas , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , 2-Hidroxipropil-beta-Ciclodextrina , Administración Intravesical , Animales , Cistitis/prevención & control , Citocinas/metabolismo , Modelos Animales de Enfermedad , Excipientes/farmacología , Femenino , Imiquimod , Ácido Láctico/farmacología , Ratones , Ratones Endogámicos C57BL , Polietilenos/farmacología , Polipropilenos/farmacología , beta-Ciclodextrinas/farmacologíaRESUMEN
Alzheimer's disease, the most common amyloid-associated disorder, accounts for the majority of the dementia diagnosed after the age of 60. The cleavage of the beta-amyloid precursor protein is initiated by beta-secretase (BACE-1), a membrane-bound aspartic protease, which has emerged as an important but difficult protein target. Here, an in silico screening approach consisting of fragment-based docking, ligand conformational search by a genetic algorithm, and evaluation of free energy of binding was used to identify low-molecular-weight inhibitors of BACE-1. More than 300,000 small molecules were docked and about 15,000 prioritized according to a linear interaction energy model with evaluation of solvation by continuum electrostatics. Eighty-eight compounds were tested in vitro, and 10 of them showed an IC(50) value lower than 100 microM in a BACE-1 enzymatic assay. Interestingly, the 10 active compounds shared a triazine scaffold. Moreover, four of them were active in an assay with mammalian cells (EC(50) < 20 microM), indicating that they are cell-permeable. Therefore, these triazine derivatives are very promising lead candidates for BACE-1 inhibition. The discoveries of this series and two other series of nonpeptidic BACE-1 inhibitors demonstrate the usefulness of our in silico high-throughput screening approach.
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Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Inhibidores de Proteasas/química , Electroquímica , Modelos Moleculares , Inhibidores de Proteasas/farmacologíaRESUMEN
The protease encoded by the human cytomegalovirus (HCMV) is an attractive target for antiviral drug development because of its essential function in viral replication. We describe here a cellular assay in the yeast Saccharomyces cerevisiae for the identification of small molecule inhibitors of HCMV protease by conditional growth in selective medium. In this system, the protease cleavage sequence is inserted into the N-(5'-phosphoribosyl)anthranilate isomerase (Trp1p), a yeast protein essential for cell proliferation in the absence of tryptophan. Coexpression of HCMV protease with the engineered Trp1p substrate in yeast cells results in site-specific cleavage and functional inactivation of the Trp1p enzyme, thereby leading to an arrest of cell proliferation. This growth arrest can be suppressed by the addition of validated HCMV protease inhibitors. The growth selection system presented here provides the basis for a high-throughput screen to identify HCMV protease inhibitors that are active in eukaryotic cells.
Asunto(s)
Isomerasas Aldosa-Cetosa/metabolismo , Antivirales/farmacología , Citomegalovirus/efectos de los fármacos , Citomegalovirus/enzimología , Inhibidores de Proteasas/farmacología , Proteínas de Saccharomyces cerevisiae/metabolismo , Isomerasas Aldosa-Cetosa/genética , Secuencia de Aminoácidos , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Datos de Secuencia Molecular , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Receptor tyrosine kinases (RTKs) play an important role in the control of fundamental cellular processes, including cell proliferation, migration, differentiation, and survival. Deregulated RTK signaling is critically involved in the development and progression of human cancer. Here, we present an assay for monitoring RTK activities in yeast, which provides an ideal heterologous cellular system to study these mammalian proteins in a null background environment. With our system, we have reconstituted aspects of the epidermal growth factor receptor (EGFR) signaling pathway as a model. Our approach is based on the Ras-recruitment system, in which membrane localization of a constitutively active human Ras achieved through protein-protein interactions can rescue growth of a temperature-sensitive yeast strain (cdc25-2). We show that co-expression of a dimerizing membrane-bound EGFR variant with specific adaptor proteins fused to the active Ras rescues growth of the cdc25-2 mutant yeast strain at the nonpermissive temperature. Using kinase-defective RTK mutants and selective EGFR kinase inhibitors, we demonstrate that growth rate of this yeast strain correlates with kinase activity of the EGFR derivatives. The RTK cellular assay presented here can be applied in high-throughput screens for selecting RTK-specific inhibitors that must be able to permeate the membrane and to function in an eukaryotic intrecellular environment.
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Biotecnología/métodos , Proteínas Fúngicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/química , Biotecnología/instrumentación , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Dimerización , Inhibidores Enzimáticos/farmacología , Receptores ErbB/metabolismo , Modelos Genéticos , Mutación , Fenotipo , Plásmidos/metabolismo , Unión Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Transducción de Señal , Temperatura , Factores de Tiempo , Proteínas ras/metabolismo , ras-GRF1/metabolismoRESUMEN
A fragment-based docking procedure followed by substructure search were used to identify active-site beta-secretase inhibitors from a composite set of about 300 000 and a library of nearly 180 000 small molecules, respectively. EC(50) values less than 10 microM were measured in at least one of two different mammalian cell-based assays for 12 of the 72 purchased compounds. In particular, the phenylureathiadiazole 2 and the diphenylurea derivative 3 are promising lead compounds for beta-secretase inhibition.
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Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/química , Carbanilidas/síntesis química , Compuestos de Fenilurea/síntesis química , Tiadiazoles/síntesis química , Secretasas de la Proteína Precursora del Amiloide , Animales , Sitios de Unión , Carbanilidas/química , Carbanilidas/farmacología , Línea Celular , Permeabilidad de la Membrana Celular , Bases de Datos Factuales , Endopeptidasas , Transferencia Resonante de Energía de Fluorescencia , Mamíferos , Modelos Moleculares , Compuestos de Fenilurea/química , Compuestos de Fenilurea/farmacología , Electricidad Estática , Relación Estructura-Actividad , Tiadiazoles/química , Tiadiazoles/farmacologíaRESUMEN
Enhancers are DNA sequences that can activate gene transcription from remote positions. In yeast, regulatory sequences that are functionally equivalent to the metazoan enhancers are called upstream activating sequences (UASs). UASs show a lower degree of flexibility than their metazoan counterparts, but can nevertheless activate transcription from a distance of >1000 bp from the promoter. One of several models for the mechanism of action of transcriptional enhancers proposes that enhancer-bound activating proteins contact promoter-bound transcription factors and thereby get in close proximity to the promoter region with concomitant looping of the intervening DNA. We tested the mode of enhancer activity in yeast. A polymerase II-transcribed gene was paired with a remote, inducible enhancer. An independent reporter system was inserted next to the promoter to monitor the potential modes of enhancer activity. Our results show that the enhancer activated the reporter system only in the presence of a functional promoter. We also demonstrate that the heterologous expression of GAGA, a factor known to facilitate DNA loop formation, allows enhancer action in yeast over a distance of 3000 bp.
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Proteínas de Unión al ADN/metabolismo , ADN/química , Proteínas de Drosophila/metabolismo , Elementos de Facilitación Genéticos , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Animales , Genes Reporteros , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
The versatile genetic malleability of yeast, and the high degree of conservation between its cellular processes and those of human cells, have made it the model of choice for pioneering research in molecular and cell biology over the past four decades. These characteristics of yeast, taken together with technical advantages such as simple growth conditions, rapid cell division and the development of a wealth of genetic tools for analysis of biological functions, have expanded the application of yeast as screening tool to the field of drug discovery.:
RESUMEN
BACKGROUND: CP-31398 is a small molecule that has been reported to stabilize the DNA-binding core domain of the human tumor suppressor protein p53 in vitro. The compound was also reported to function as a potential anti-cancer drug by rescuing the DNA-binding activity and, consequently, the transcription activation function of mutant p53 protein in mammalian tissue culture cells and in mice. RESULTS: We performed a series of gene expression experiments to test the activity of CP-31398 in yeast and in human cell cultures. With these cell-based assays, we were unable to detect any specific stimulation of mutant p53 activity by this compound. Concentrations of CP-31398 that were reported to be active in the published work were highly toxic to the human H1299 lung carcinoma and Saos-2 cell lines in our experiments. CONCLUSION: In our experiments, the small molecule CP-31398 was unable to reactivate mutant p53 protein. The results of our in vivo experiments are in agreement with the recently published biochemical analysis of CP-31398 showing that this molecule does not bind p53 as previously claimed, but intercalates into DNA.
Asunto(s)
Pirimidinas/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Activación Transcripcional/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Daunorrubicina/farmacología , Genes Reporteros , Humanos , Saccharomyces cerevisiae/citologíaRESUMEN
The reverse two-hybrid system has been developed to readily identify molecules or mutations that can disrupt protein-protein interactions in vivo. This system is generally based on the interaction-dependent activation of a reporter gene, whose product inhibits the growth of the engineered yeast cell. Thus, disruption of the interaction between the hybrid proteins can be positively selected because, by reducing the expression of the negative marker gene, it allows cell growth. Although several counter-selectable marker genes are currently available, their application in the reverse two-hybrid system is generally confronted with technical and practical problems such as low selectivity and relatively complex experimental procedures. Thus, the characterization of more reliable and simple counter-selection assays for the reverse two-hybrid system continues to be of interest. We have developed a novel counter-selection assay based on the toxicity of intracellular galactose-1-phosphate, which accumulates upon expression of a galactokinase-encoding GAL1 reporter gene in the absence of transferase activity. Decreased GAL1 gene expression upon dissociation of interacting proteins causes reduction of intracellular galactose-1-phosphate concentrations, thus allowing cell growth under selective conditions.
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Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Galactoquinasa/genética , Galactoquinasa/metabolismo , Galactosafosfatos/metabolismo , Ingeniería de Proteínas/métodos , Mapeo de Interacción de Proteínas/métodos , Levaduras/fisiología , Técnicas del Sistema de Dos HíbridosRESUMEN
Abeta peptides, which are believed to be at the origin of Alzheimer's disease (AD), are produced through the sequential processing of the transmembrane amyloid precursor protein (APP) by the beta- and gamma-secretase. The identification of small molecules that penetrate the brain and inhibit these secretases is of great therapeutic potential. Here, we describe a cellular selection system in yeast for the identification of inhibitors of the human beta-secretase BACE-1. Similar to the natural situation in mammalian cells, BACE-1 and its substrate APP are bound to membranes in secretory pathway compartments. Yeast cells expressing these human proteins have been engineered so as to grow under selective conditions only upon reduction of BACE-1 activity, thus allowing identification of compounds that, in addition to inhibiting BACE-1, must permeate cellular membranes and present no cytotoxic effects. Our results show that gradual reduction of BACE-1 expression in the engineered yeast strain resulted in gradual increase of cell growth rate. Moreover, two validated BACE-1 inhibitors, which have IC50 values between 7 and 8 microM in mammalian cell assays, stimulated yeast growth in a concentration-dependent manner. This effect was specific for BACE-1 since these compounds had no effect on yeast cells expressing a different secretase cleaving the APP substrate at the alpha-site. The target-specific cellular assay presented here is applicable in high-throughput screens for selecting inhibitors of defined secretases acting on natural substrates in a membrane-bound protein configuration.
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Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/metabolismo , Inhibidores de Proteasas/metabolismo , Levaduras/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Ácido Aspártico Endopeptidasas/genética , Bioensayo , División Celular/fisiología , Doxiciclina/farmacología , Endopeptidasas , Regulación de la Expresión Génica , Humanos , Regiones Promotoras Genéticas , Transcripción Genética , Levaduras/efectos de los fármacos , Levaduras/genéticaRESUMEN
Plant lateral organs exhibit proximal-distal and adaxial-abaxial polarity. In Arabidopsis, abaxial cell fate is regulated in part by putative transcription factors of the YABBY family, such as FILAMENTOUS FLOWER (FIL) and INNER NO OUTER (INO), by a mechanism that currently is not fully understood. NOZZLE (NZZ) encodes a plant-specific nuclear protein. Genetic evidence has shown that NZZ is involved in the positive feedback regulation of INO, thereby acting both as a temporal and spatial repressor of INO transcription. This mechanism allows the ovule primordium to complete its proximal-distal organization, prior to the onset of adaxial-abaxial development in the chalaza. During our study, we isolated FIL in a yeast two-hybrid screen using NZZ as bait. In vitro pull-down experiments confirmed the NZZ-FIL interaction. NZZ also bound INO and YABBY3, suggesting that NZZ generally interacts with YABBY proteins in vitro. The polar-charged region of NZZ was necessary and sufficient to bind to the zinc finger of INO and to interact with its C terminus carrying the high mobility group-like domain. We suggest that NZZ coordinates proximal-distal patterning and adaxial-abaxial polarity establishment in the developing ovule by directly binding to INO.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/anatomía & histología , Arabidopsis/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Cartilla de ADN , Flores/metabolismo , Flores/ultraestructura , Proteínas Nucleares/genética , Plásmidos/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Dedos de ZincRESUMEN
The intracellular expression of highly specific antibody fragments ("intrabodies") in eukaryotes has a great potential in functional genomics and therapeutics. However, since the intracellular reducing environment prevents formation of the conserved intrachain disulfide bonds, most antibodies do not fold properly and are therefore inactive inside cells. The few antibodies that have been found to function in an intracellular environment and that have been characterized for their biophysical properties have generally shown a high degree of stability and solubility. Thus, for intracellular expression and application, very stable antibody frameworks are needed that can correctly fold even in the absence of disulfide bonds and that do not aggregate. Here, we present and discuss a novel method, named "Quality Control," which allows selection of stable and soluble antibody frameworks in vivo without the requirement or knowledge of antigens. This system is based on the expression of single-chain antibody fragments (scFvs) fused to a selectable marker that can control gene expression and cell growth. The activity of such a selectable marker fused to various scFvs that have been biophysically characterized correlated with the solubility and stability of the scFv moieties. This antigen-independent intrabody selection system was applied to screen scFv libraries for identifying stable and soluble frameworks, which subsequently served as acceptor backbones to construct intrabody libraries by randomization of hypervariable loops.
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Anticuerpos/química , Antígenos/inmunología , Líquido Intracelular/química , Anticuerpos/genética , Antígenos/química , Antígenos/genética , Línea Celular , Regulación de la Expresión Génica , Terapia Genética/métodos , Humanos , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/inmunologíaRESUMEN
The beta-secretase BACE1 is an attractive drug target for reducing the level of the Alzheimer's disease-promoting Abeta peptide in the brain. Whereas potent peptidomimetic in vitro inhibitors of BACE1 have been designed, screening approaches to identify cell-permeable small molecule inhibitors have had limited success so far. In the present minireview we summarize existing screening methods, discuss their scope of application in the drug discovery process and compare them to a novel cell-based screening system to identify BACE1 inhibitors by a positive yeast growth selection.
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Ácido Aspártico Endopeptidasas/efectos de los fármacos , Endopeptidasas/efectos de los fármacos , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Enfermedad de Alzheimer/enzimología , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , División Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Endopeptidasas/genética , Endopeptidasas/metabolismo , Inhibidores de Proteasas/metabolismo , Levaduras/efectos de los fármacos , Levaduras/genética , Levaduras/metabolismoRESUMEN
The interplay among extracellular and cell surface proteins, such as the interactions between ligands and receptors or between antigens and antibodies, is involved in a multitude of physiological and pathological phenomena. In the oxidizing milieu of the secretory pathway in eukaryotic cells, many extracellular proteins build disulfide bonds that significantly contribute to their correct folding and structural stability. Thus, conventional yeast two-hybrid interaction assays, which occur in the reducing intracellular environment, might not be adequate to detect extracellular protein-protein interactions. We have exploited the properties of yeast Ire1p, a type I endoplasmic reticulum (ER) membrane protein involved in the unfolded protein response (UPR) as a dimerization-activated receptor, to develop a novel system for the detection and study of interactions between extracellular and/or membrane proteins. In our system, named SCINEX-P (screening for interactions between extracellular proteins), proteins of interest were fused to truncated Ire1p so as to substitute its N-terminal lumenal domain (NLD). Specific interaction between two partners caused dimerization of the Ire1p moiety, which, through the endogenous UPR signalling pathway, led to activation of transcription of genes that permit cell growth under selective conditions.
Asunto(s)
División Celular , Ensayo de Unidades Formadoras de Colonias/métodos , Glicoproteínas de Membrana/química , Proteínas de la Membrana/química , Proteínas Serina-Treonina Quinasas/química , Proteínas de Saccharomyces cerevisiae/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Dimerización , Activación Enzimática , Regulación de la Expresión Génica , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Pliegue de Proteína , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Proteínas Represoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Temperatura , Factores de Transcripción/genéticaRESUMEN
Sequential processing of the transmembrane amyloid precursor protein (APP) by the beta-secretase BACE and by the gamma-secretase causes secretion of Abeta peptides. Extracellular aggregation of these peptides in the brain is a major hallmark of Alzheimer's disease. For therapeutic purposes and the development of specific inhibitors, it is important to characterize these secretases. We have established a cellular growth selection system for functional expression of human BACE in the yeast Saccharomyces cerevisiae. A fragment of APP bearing the beta-site, the transmembrane domain and the cytosolic tail was fused to the C-terminus of the yeast enzyme invertase, which is normally secreted to allow cell growth in the presence of sucrose as the sole carbon source. The resulting invertase-APP fusion protein was expressed as a type-I transmembrane protein in intracellular compartments of yeast cells lacking endogenous invertase. In these cells, co-expression of human BACE restored cell growth on selective plates upon cleavage of the invertase-APP fusion protein. The cellular growth selection system presented here can be generally applied to screen for secretases that specifically cleave membrane-bound substrates. Furthermore, this system provides the basis for a high-throughput screen for identifying secretase inhibitors that are active in eukaryotic cells.