RESUMEN
The androgen receptor (AR) is an important target for drug therapies combating prostate cancer. However, various acquired mutations within the AR sequence often render this receptor resistant to treatment. Ligand-induced interaction between the N- and C-termini of the AR marks the initial step in the AR signaling cascade and can thus serve as an early read-out for analysis of potential antagonists of wt and mutant AR. To measure changes of the N/C interaction in the wt and mutant AR variants upon the addition of inhibitors, we applied our recently developed Fluorescent Two-Hybrid (F2H) assay. The F2H method enables real-time monitoring and quantitative analysis of the interactions between GFP- and RFP-tagged proteins in live mammalian cells, where GFP-tagged proteins are tethered to a specific nuclear location. This anchoring approach provides a local signal enrichment suitable for direct visualization of protein-protein interactions as co-localizations by conventional epifluorescence microscopy. Since the F2H assay is fully reversible, we could monitor dynamics of AR N/C interactions in living cells in real time upon agonistic, as well as antagonistic treatments. In dose-response F2H experiments, we compared the potencies of abiraterone, bicalutamide, enzalutamide, flutamide, and galeterone/TOK-001 to prevent the dihydrotestosterone-induced N/C interaction in wt AR. We further applied the newly developed F2H assay to analyze how the AR N/C interaction is affected by the clinically relevant mutations W741L, F876L, T877A and F876L/T877A. We conclude that F2H is a reliable and technically undemanding approach for straightforward screening of new AR modulators, as well as for monitoring their activity in real time in living cells.
Asunto(s)
Antagonistas de Andrógenos/química , Andrógenos/química , Microscopía Fluorescente/métodos , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Androstadienos/química , Androstenos/química , Anilidas/química , Animales , Benzamidas , Bencimidazoles/química , Bioensayo , Línea Celular , Cricetinae , Dihidrotestosterona/química , Flutamida/química , Células HEK293 , Humanos , Masculino , Mutación , Nitrilos/química , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/química , Neoplasias de la Próstata/genética , Compuestos de Tosilo/química , Factores de Transcripción/antagonistas & inhibidores , Técnicas del Sistema de Dos HíbridosRESUMEN
Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (~ 15 kDa), termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair.
Asunto(s)
Poli(ADP-Ribosa) Polimerasas/metabolismo , Anticuerpos de Dominio Único/inmunología , Resonancia por Plasmón de Superficie/métodos , Especificidad de Anticuerpos , Línea Celular , Supervivencia Celular , ADN/genética , ADN/metabolismo , Epítopos/inmunología , Humanos , Inmunoprecipitación , Imagen Molecular , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/inmunología , Estructura Terciaria de Proteína , Transporte de ProteínasRESUMEN
Protein-protein interactions (PPIs) are attractive but challenging targets for drug discovery. To overcome numerous limitations of the currently available cell-based PPI assays, we have recently established a fully reversible microscopy-assisted fluorescent two-hybrid (F2H) assay. The F2H assay offers a fast and straightforward readout: an interaction-dependent co-localization of two distinguishable fluorescent signals at a defined spot in the nucleus of mammalian cells. We developed two reversible F2H assays for the interactions between the tumor suppressor p53 and its negative regulators, Mdm2 and Mdm4. We then performed a pilot F2H screen with a subset of compounds, including small molecules (such as Nutlin-3) and stapled peptides. We identified five cell-penetrating compounds as potent p53-Mdm2 inhibitors. However, none exhibited intracellular activity on p53-Mdm4. Live cell data generated by the F2H assays enable the characterization of stapled peptides based on their ability to penetrate cells and disrupt p53-Mdm2 interaction as well as p53-Mdm4 interaction. Here, we show that the F2H assays enable side-by-side analysis of substances' dual Mdm2-Mdm4 activity. In addition, they are suitable for testing various types of compounds (e.g., small molecules and peptidic inhibitors) and concurrently provide initial data on cellular toxicity. Furthermore, F2H assays readily allow real-time visualization of PPI dynamics in living cells.
Asunto(s)
Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Técnica del Anticuerpo Fluorescente , Unión Proteica/efectos de los fármacos , Técnicas del Sistema de Dos Híbridos , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cricetinae , Humanos , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Pharmacological modulation of p53 activity is an attractive therapeutic strategy in cancers with wild-type p53. Presently in clinical trials, the small molecule Nutlin-3A competitively binds to HDM2, a key negative regulator of p53 and blocks its activity. We have described resistance mutations in HDM2 that selectively reduce affinity for Nutlin but not p53. In the present communication, we show that stapled peptides targeting the same region of HDM2 as Nutlin are refractory to these mutations, and display reduced discrimination between the wild-type and mutant HDM2s with regards to functional abrogation of interaction with p53. The larger interaction footprint afforded by stapled peptides suggests that this class of ligands may prove comparatively more resilient to acquired resistance in a clinical setting.
Asunto(s)
Resistencia a Antineoplásicos/genética , Imidazoles/farmacología , Mutación , Péptidos/farmacología , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/genética , Animales , Línea Celular , Técnicas de Inactivación de Genes , Humanos , Ligandos , Ratones , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Neoplasias/genética , Péptidos/química , Péptidos/metabolismo , Unión Proteica/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
HDM2 binds to the p53 tumour suppressor and targets it for proteosomal degradation. Presently in clinical trials, the small molecule Nutlin-3A competitively binds to HDM2 and abrogates its repressive function. Using a novel in vitro selection methodology, we simulated the emergence of resistance by evolving HDM2 mutants capable of binding p53 in the presence of Nutlin concentrations that inhibit the wild-type HDM2-p53 interaction. The in vitro phenotypes were recapitulated in ex vivo assays measuring both p53 transactivation function and the direct p53-HDM2 interaction in the presence of Nutlin. Mutations conferring drug resistance were not confined to the N-terminal p53/Nutlin-binding domain, and were additionally seen in the acidic, zinc finger and RING domains. Mechanistic insights gleaned from this broad spectrum of mutations will aid in future drug design and further our understanding of the complex p53-HDM2 interaction.
Asunto(s)
Antineoplásicos/química , Imidazoles/química , Simulación del Acoplamiento Molecular , Mutación , Piperazinas/química , Proteínas Proto-Oncogénicas c-mdm2/química , Proteína p53 Supresora de Tumor/química , Animales , Antineoplásicos/farmacología , Sitios de Unión , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Expresión Génica , Biblioteca de Genes , Humanos , Imidazoles/farmacología , Ratones , Piperazinas/farmacología , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
By using a phage display derived peptide as an initial template, compounds have been developed that are highly specific against Mdm2/Mdm4. These compounds exhibit greater potency in p53 activation and protein-protein interaction assays than a compound derived from the p53 wild-type sequence. Unlike Nutlin, a small molecule inhibitor of Mdm2/Mdm4, the phage derived compounds can arrest cells resistant to p53 induced apoptosis over a wide concentration range without cellular toxicity, suggesting they are highly suitable for cyclotherapy.
Asunto(s)
Péptidos/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de Ciclo Celular , Humanos , Modelos Moleculares , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Péptidos/química , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/químicaRESUMEN
Different bioactive molecules are released into living cells from lipid-covered mesoporous silica nanoparticles. The release is triggered by light, as the particles feature covalently attached photosensitizers as membrane-opening agents. It is demonstrated that the particles achieve endosomal escape and that they release their cargo into the cytosol.
Asunto(s)
Nanocápsulas/química , Nanocápsulas/efectos de la radiación , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/efectos de la radiación , Protoporfirinas/química , Dióxido de Silicio/sangre , Dióxido de Silicio/efectos de la radiación , Luz , Ensayo de Materiales , Nanocápsulas/ultraestructura , Tamaño de la Partícula , Porosidad/efectos de la radiación , Protoporfirinas/efectos de la radiaciónRESUMEN
The understanding of cellular processes and their physiopathological alterations requires comprehensive data on the abundance, distribution, modification and interaction of cellular components. On the one hand, artificially introduced fluorescent fusion proteins provide information about their distribution and dynamics in living cells but not on endogenous factors. On the other hand, antibodies can detect endogenous proteins, posttranslational modifications and other cellular components but mostly in fixed and permeabilized cells. Here we highlight a new technology based on the antigen-binding domain of heavy-chain antibodies (VHH) from Camelidae. We have demonstrated that these VHH domains can be fused with fluorescent proteins and expressed in living cells. Those fluorescent antigen-binding proteins-called chromobodies-can be used to detect and trace proteins and other cellular components in vivo. In principle chromobodies can detect any antigenic structure including posttranslational modifications or nonprotein components and thereby dramatically expand the quality and quantity of information that can be gathered in high-content analyses. Here we demonstrate the suitability of this technology to follow apoptosis in living cells in real time.
Asunto(s)
Apoptosis/fisiología , Imagen Molecular/métodos , Anticuerpos de Dominio Único , Línea Celular , HumanosRESUMEN
Camel single-domain antibody fragments or Nanobodies, are practical in a wide range of applications. Their unique biochemical and biophysical properties permit an intracellular expression and antigen targeting. The availability of an efficient intracellular selection step would immediately identify the best intracellularly performing functional antibody fragments. Therefore, we assessed a bacterial-two-hybrid system to retrieve such Nanobodies. With GFP as an antigen we demonstrate that antigen-specific Nanobodies of sub-micromolar affinity and stability above 30 kJ/mol, at a titer of 10(-4) can be retrieved in a single-step selection. This was further proven practically by the successful recovery from an 'immune' library of multiple stable, antigen-specific Nanobodies of good affinity for HIV-1 integrase or nucleoside hydrolase. The sequence diversity, intrinsic domain stability, antigen-specificity and affinity of these binders compare favorably to those that were retrieved in parallel by phage display pannings.
Asunto(s)
Camelus/inmunología , Clonación Molecular/métodos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos , Camelus/genética , Línea Celular , Escherichia coli/genética , Expresión Génica , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Integrasa de VIH/inmunología , VIH-1/enzimología , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/inmunología , Biblioteca de Péptidos , Estabilidad Proteica , Trypanosoma vivax/enzimologíaRESUMEN
Information about protein interactions is crucial for the understanding of cellular processes. Current methods for the investigation of protein-protein interactions (PPIs) require either removal of the proteins from their normal cellular environment, perturbation of the cells or costly instrumentation and advanced technical expertise (Fields and Song, Nature 340:245-246, 1989; Deane et al., Mol Cell Proteomics 1:349-356, 2002; Kerppola, Nat Rev Mol Cell Biol 7:449-456, 2006; Blanchard et al., Mol Cell Proteomics 5:2175-2184, 2006; Miller et al., Mol Cell Proteomics 6:1027-1038, 2007; Miyawaki, Dev Cell 4:295-305, 2003; Parrish et al., Curr Opin Biotechnol 17:387-393, 2006; Sekar and Periasamy, J Cell Biol 160:629-633, 2003). Here, we describe a simple assay to directly visualize and analyze PPIs in single living cells. By adapting a lac operator/repressor system, we generated a stable nuclear interaction platform. A fluorescent bait protein is tethered to the interaction platform and assayed for co-localization of fluorescent prey fusion proteins. This fluorescent two-hybrid (F2H) assay allows the investigation of cell cycle dependent PPIs. With this cell based assay protein interactions even from different subcellular compartments can be visualized in real time (Zolghadr et al., Mol Cell Proteomics 7:2279-2287, 2008). The simple optical readout enables automated imaging systems to segment and analyze the acquired data for high-throughput screening of PPIs in living cells in response to external stimuli and chemical compounds.
Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Proteínas/análisis , Animales , Línea Celular , Supervivencia Celular , Clonación Molecular , Cricetinae , Humanos , Microscopía , Proteínas/metabolismo , Espectrometría de Fluorescencia , TransfecciónRESUMEN
The understanding of cellular processes and their pathophysiological alterations requires comprehensive data on the abundance, distribution, modification, and interaction of all cellular components. On the one hand, artificially introduced fluorescent fusion proteins provide information about their distribution and dynamics in living cells but not about endogenous factors. On the other hand, antibodies can detect endogenous proteins, posttranslational modifications, and other cellular components but mostly in fixed and permeabilized cells. Here we highlight a new technology based on the antigen-binding domain of heavy-chain antibodies (V(H)H) from Camelidae. These extremely stable V(H)H domains can be produced in bacteria, coupled to matrices, and used for affinity purification and proteome studies. Alternatively, these V(H)H domains can be fused with fluorescent proteins and expressed in living cells. These fluorescent antigen-binding proteins called "chromobodies" can be used to detect and trace proteins and other cellular components in vivo. Chromobodies can, in principle, detect any antigenic structure, including posttranslational modifications, and thereby dramatically expand the quality and quantity of information that can be gathered in high-content analysis. Depending on the epitope chosen, chromobodies can also be used to modulate protein function in living cells.
Asunto(s)
Anticuerpos/química , Técnica del Anticuerpo Fluorescente/métodos , Proteínas/química , Proteoma/química , Animales , Anticuerpos/genética , Anticuerpos/metabolismo , Línea Celular , Células/química , Células/metabolismo , Humanos , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Conformación Proteica , Proteínas/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Genetic high throughput screens have yielded large sets of potential protein-protein interactions now to be verified and further investigated. Here we present a simple assay to directly visualize protein-protein interactions in single living cells. Using a modified lac repressor system, we tethered a fluorescent bait at a chromosomal lac operator array and assayed for co-localization of fluorescent prey fusion proteins. With this fluorescent two-hybrid assay we successfully investigated the interaction of proteins from different subcellular compartments including nucleus, cytoplasm, and mitochondria. In combination with an S phase marker we also studied the cell cycle dependence of protein-protein interactions. These results indicate that the fluorescent two-hybrid assay is a powerful tool to investigate protein-protein interactions within their cellular environment and to monitor the response to external stimuli in real time.
Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Técnicas del Sistema de Dos Híbridos , Animales , Secuencia de Bases , Ciclo Celular , Línea Celular , Cricetinae , Cartilla de ADN/genética , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Operón Lac , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteómica/métodos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , TransfecciónRESUMEN
Green fluorescent proteins (GFPs) and variants thereof are widely used to study protein localization and dynamics. We engineered a specific binder for fluorescent proteins based on a 13-kDa GFP binding fragment derived from a llama single chain antibody. This GFP-binding protein (GBP) can easily be produced in bacteria and coupled to a monovalent matrix. The GBP allows a fast and efficient (one-step) isolation of GFP fusion proteins and their interacting factors for biochemical analyses including mass spectroscopy and enzyme activity measurements. Moreover GBP is also suitable for chromatin immunoprecipitations from cells expressing fluorescent DNA-binding proteins. Most importantly, GBP can be fused with cellular proteins to ectopically recruit GFP fusion proteins allowing targeted manipulation of cellular structures and processes in living cells. Because of the high affinity capture of GFP fusion proteins in vitro and in vivo and a size in the lower nanometer range we refer to the immobilized GFP-binding protein as GFP-nanotrap. This versatile GFP-nanotrap enables a unique combination of microscopic, biochemical, and functional analyses with one and the same protein.
Asunto(s)
Proteínas Luminiscentes/metabolismo , Nanotecnología/métodos , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Núcleo Celular/metabolismo , Inmunoprecipitación de Cromatina , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/aislamiento & purificación , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Proteínas Luminiscentes/química , Proteínas Luminiscentes/aislamiento & purificación , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
We fused the epitope-recognizing fragment of heavy-chain antibodies from Camelidae sp. with fluorescent proteins to generate fluorescent, antigen-binding nanobodies (chromobodies) that can be expressed in living cells. We demonstrate that chromobodies can recognize and trace antigens in different subcellular compartments throughout S phase and mitosis. Chromobodies should enable new functional studies, as potentially any antigenic structure can be targeted and traced in living cells in this fashion.
Asunto(s)
Anticuerpos/química , Antígenos/química , Camelus/inmunología , Animales , Anticuerpos/inmunología , Reacciones Antígeno-Anticuerpo , Antígenos/inmunología , Epítopos , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/inmunología , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/inmunología , Mitosis , Fase SRESUMEN
DNA methyltransferases have a central role in the complex regulatory network of epigenetic modifications controlling gene expression in mammalian cells. To study the regulation of DNA methylation in living cells, we developed a trapping assay using transiently expressed fluorescent DNA methyltransferase 1 (Dnmt1) fusions and mechanism-based inhibitors 5-azacytidine (5-aza-C) or 5-aza-2'-deoxycytidine (5-aza-dC). These nucleotide analogs are incorporated into the newly synthesized DNA at nuclear replication sites and cause irreversible immobilization, that is, trapping of Dnmt1 fusions at these sites. We measured trapping by either fluorescence bleaching assays or photoactivation of photoactivatable green fluorescent protein fused to Dnmt1 (paGFP-Dnmt1) in mouse and human cells; mutations affecting the catalytic center of Dnmt1 prevented trapping. This trapping assay monitors kinetic properties and activity-dependent immobilization of DNA methyltransferases in their native environment, and makes it possible to directly compare mutations and inhibitors that affect regulation and catalytic activity of DNA methyltransferases in single living cells.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/análisis , Metilación de ADN , Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Animales , Azacitidina/análogos & derivados , Azacitidina/farmacología , Núcleo Celular/enzimología , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Replicación del ADN , Decitabina , Proteínas Fluorescentes Verdes/análisis , Humanos , Ratones , Fotoquímica/métodos , Proteínas Recombinantes de Fusión/análisisRESUMEN
Previous work has demonstrated a more decondensed large-scale chromatin structure and a more internal nuclear position for gene-rich versus gene-poor chromosome regions. Here, we show that large-scale chromatin opening and changes in intranuclear positioning of chromosome regions can be induced by normal levels of endogenous transcription factors acting on mammalian regulatory sequences. We transfected mouse erythroleukemia cells with a 15 kbp plasmid containing a lac operator repeat plus beta-globin regulatory sequences driving a beta-galactosidase reporter gene. After green-fluorescent-protein/lac-repressor fusion-protein binding or after fluorescence in situ hybridization, the volume and location of the transgene array signal were measured. With both detection methods, we found that the volume was severalfold larger when transcription was on. While silent transgene arrays were located close to the nuclear membrane, we observed a significantly more internal position for the transcriptionally active state. Our results indicate that both large-scale chromatin decondensation and changes in nuclear positioning as observed for large, complex gene-rich chromosome regions can be reproduced by endogenous regulatory sequences acting within simple repetitive transgene arrays.