RESUMEN
The MRE11 nuclease is essential during DNA damage recognition, homologous recombination, and replication. BRCA2 plays important roles during homologous recombination and replication. Here, we show that effecting an MRE11 blockade using a prototypical inhibitor (Mirin) induces synthetic lethality (SL) in BRCA2-deficient ovarian cancer cells, HeLa cells, and 3D spheroids compared to BRCA2-proficient controls. Increased cytotoxicity was associated with double-strand break accumulation, S-phase cell cycle arrest, and increased apoptosis. An in silico analysis revealed Mirin docking onto the active site of MRE11. While Mirin sensitises DT40 MRE11+/- cells to the Top1 poison SN-38, it does not sensitise nuclease-dead MRE11 cells to this compound confirming that Mirin specifically inhibits Mre11 nuclease activity. MRE11 knockdown reduced cell viability in BRCA2-deficient PEO1 cells but not in BRCA2-proficient PEO4 cells. In a Mirin-resistant model, we show the downregulation of 53BP1 and DNA repair upregulation, leading to resistance, including in in vivo xenograft models. In a clinical cohort of human ovarian tumours, low levels of BRCA2 expression with high levels of MRE11 co-expression were linked with worse progression-free survival (PFS) (p = 0.005) and overall survival (OS) (p = 0.001). We conclude that MRE11 is an attractive SL target, and the pharmaceutical development of MRE11 inhibitors for precision oncology therapeutics may be of clinical benefit.
Asunto(s)
Proteínas de Unión al ADN , Neoplasias Ováricas , Humanos , Femenino , Proteínas de Unión al ADN/metabolismo , Proteína Homóloga de MRE11/genética , Proteína Homóloga de MRE11/metabolismo , Células HeLa , Medicina de Precisión , Proteína BRCA2/metabolismo , Reparación del ADN , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Línea Celular TumoralRESUMEN
The screening of compound libraries to identify small-molecule modulators of specific biological targets is crucial in the process for the discovery of novel therapeutics and molecular probes. Considering the need for simple single-tool assay technologies with which one could monitor "all" kinases, we developed a fluorescence polarization (FP)-based assay to monitor the binding capabilities of protein kinases to ATP. We used BODIPY ATP-y-S as a probe to measure the shift in the polarization of a light beam when passed through the sample. We were able to optimize the assay using commercial Protein Kinase A (PKA) and H7 efficiently inhibited the binding of the probe when added to the reaction. Furthermore, we were able to employ the assay in a high-throughput fashion and validate the screening of a set of small molecules predicted to dock into the ATP-binding site of PKA. This will be useful to screen larger libraries of compounds that may target protein kinases by blocking ATP binding.
RESUMEN
Coagulation factor XII (FXII) is a key initiator of the contact pathway, which contributes to inflammatory pathways. FXII circulates as a zymogen, which when auto-activated forms factor XIIa (FXIIa). Here, the production of the recombinant FXIIa protease domain (ßFXIIaHis) with yields of â¼1-2â mg per litre of insect-cell culture is reported. A second construct utilized an N-terminal maltose-binding protein (MBP) fusion (MBP-ßFXIIaHis). Crystal structures were determined of MBP-ßFXIIaHis in complex with the inhibitor D-Phe-Pro-Arg chloromethyl ketone (PPACK) and of ßFXIIaHis in isolation. The ßFXIIaHis structure revealed that the S2 and S1 pockets were occupied by Thr and Arg residues, respectively, from an adjacent molecule in the crystal. The Thr-Arg sequence mimics the P2-P1 FXIIa cleavage-site residues present in the natural substrates prekallikrein and FXII, and Pro-Arg (from PPACK) mimics the factor XI cleavage site. A comparison of the ßFXIIaHis structure with the available crystal structure of the zymogen-like FXII protease revealed large conformational changes centred around the S1 pocket and an alternate conformation for the 99-loop, Tyr99 and the S2 pocket. Further comparison with activated protease structures of factors IXa and Xa, which also have the Tyr99 residue, reveals that a more open form of the S2 pocket only occurs in the presence of a substrate mimetic. The FXIIa inhibitors EcTI and infestin-4 have Pro-Arg and Phe-Arg P2-P1 sequences, respectively, and the interactions that these inhibitors make with ßFXIIa are also described. These structural studies of ßFXIIa provide insight into substrate and inhibitor recognition and establish a scaffold for the structure-guided drug design of novel antithrombotic and anti-inflammatory agents.
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Factor XIIa , Proteínas de Unión a Maltosa , Proteínas Recombinantes de Fusión/química , Clorometilcetonas de Aminoácidos/química , Animales , Sitios de Unión , Línea Celular , Cristalización , Cristalografía por Rayos X/métodos , Drosophila melanogaster , Factor XIIa/química , Factor XIIa/metabolismo , Proteínas de Unión a Maltosa/química , Proteínas de Unión a Maltosa/metabolismo , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Especificidad por SustratoRESUMEN
The AnxA2/S100A10 complex has been implicated in various placental functions but although the localisation of these proteins individually has been studied, there is no information about the localisation of their complex in situ at the cellular level. Using the proximity ligation technique, we have investigated the in situ localisation of AnxA2/S100A10 complex in the placenta and have compared this with the location patterns of the individual proteins. High levels of expression of AnxA2/S100A10 complexes were observed in the amniotic membrane and in blood vessel endothelial cells. Lower levels were detected in the brush border area of the syncytium and in the trophoblasts. Immunohistochemical analysis of AnxA2 and S100A10 individually revealed broadly similar patterns of localisation. The brush border staining pattern suggests that in this location at least some of the AnxA2 is not in complex with S100A10. The formal location of the AnxA2/S100A10 complex is compatible with a role in cell-cell interaction, intracellular transport and secretory processes and regulation of cell surface proteases, implying contributions to membrane integrity, nutrient exchange, placentation and vascular remodelling in different parts of the placenta. Future applications will allow specific assessment of the association of the complex with pathophysiological disorders.
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Anexina A2/análisis , Complejos Multiproteicos/análisis , Proteínas S100/análisis , Amnios/metabolismo , Biomarcadores/análisis , Células Endoteliales/metabolismo , Femenino , Humanos , Inmunohistoquímica , Placenta/metabolismo , Embarazo , Proteínas Gestacionales/análisis , Unión Proteica , Trofoblastos/metabolismoRESUMEN
Increasing evidence suggests that cancer cell populations contain a small proportion of cells that display stem-like cell properties and which may be responsible for overall tumor maintenance. These cancer stem-like cells (CSCs) appear to have unique tumor-initiating ability and innate survival mechanisms that allow them to resist cancer therapies, consequently promoting relapses. Selective targeting of CSCs may provide therapeutic benefit and several recent reports have indicated this may be possible. In this article, we review drugs targeting CSCs, in selected epithelial cell-derived cancers. Stem Cells 2017;35:839-850.
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Antineoplásicos/uso terapéutico , Células Epiteliales/patología , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Células Madre Neoplásicas/patología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Células Epiteliales/efectos de los fármacos , Humanos , Células Madre Neoplásicas/efectos de los fármacosRESUMEN
During sexual transmission of human immunodeficiency virus (HIV), macrophages are initial targets for HIV infection. Secretory leukocyte protease inhibitor (SLPI) has been shown to protect against HIV infection of macrophages through interactions with annexin A2 (A2), which is found on the macrophage cell surface as a heterotetramer (A2t) consisting of A2 and S100A10. Therefore, we investigated potential protein-protein interactions between A2 and HIV-1 gp120 through a series of co-immunoprecipitation assays and a single molecule pulldown (SiMPull) technique. Additionally, inhibitors of A2t (A2ti) that target the interaction between A2 and S100A10 were tested for their ability to impair productive HIV-1 infection of macrophages. Our data suggest that interactions between HIV-1 gp120 and A2 exist, though this interaction may be indirect. Furthermore, an anti-A2 antibody impaired HIV-1 particle production in macrophages in vitro, whereas A2ti did not indicating that annexin A2 may promote HIV-1 infection of macrophages in its monomeric rather than tetrameric form.
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Anexina A2/antagonistas & inhibidores , VIH-1/inmunología , VIH-1/fisiología , Interacciones Huésped-Patógeno , Macrófagos/virología , Replicación Viral , Anexina A2/metabolismo , Anticuerpos/metabolismo , Centrifugación , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Inmunoprecipitación , Unión Proteica , Mapeo de Interacción de ProteínasRESUMEN
The natural product staurosporine is a high-affinity inhibitor of nearly all mammalian protein kinases. The labelling of staurosporine has proven effective as a means of generating protein kinase research tools. Most tools have been generated by acylation of the 4'-methylamine of the sugar moiety of staurosporine. Herein we describe the alkylation of this group as a first step to generate a fluorescently labelled staurosporine. Following alkylation, a polyethylene glycol linker was installed, allowing subsequent attachment of fluorescein. We report that this fluorescein-staurosporine conjugate binds to cAMP-dependent protein kinase in the nanomolar range. Furthermore, its binding can be antagonised with unmodified staurosporine as well as ATP, indicating it targets the ATP binding site in a similar fashion to native staurosporine. This reagent has potential application as a screening tool for protein kinases of interest.
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Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Colorantes Fluorescentes/química , Inhibidores de Proteínas Quinasas/metabolismo , Estaurosporina/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Alquilación , Sitios de Unión , Proteínas Quinasas Dependientes de AMP Cíclico/química , Polarización de Fluorescencia , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Estaurosporina/síntesis química , Estaurosporina/metabolismoRESUMEN
Annexin A2 (AnxA2) was originally identified as a substrate of the pp60v-src oncoprotein in transformed chicken embryonic fibroblasts. It is an abundant protein that associates with biological membranes as well as the actin cytoskeleton, and has been implicated in intracellular vesicle fusion, the organization of membrane domains, lipid rafts and membrane-cytoskeleton contacts. In addition to an intracellular role, AnxA2 has been reported to participate in processes localized to the cell surface including extracellular protease regulation and cell-cell interactions. There are many reports showing that AnxA2 is differentially expressed between normal and malignant tissue and potentially involved in tumour progression. An important aspect of AnxA2 function relates to its interaction with small Ca(2+) -dependent adaptor proteins called S100 proteins, which is the topic of this review. The interaction between AnxA2 and S100A10 has been very well characterized historically; more recently, other S100 proteins have been shown to interact with AnxA2 as well. The biochemical evidence for the occurrence of these protein interactions will be discussed, as well as their function. Recent studies aiming to generate inhibitors of S100 protein interactions will be described and the potential of these inhibitors to further our understanding of AnxA2 S100 protein interactions will be discussed.
Asunto(s)
Anexina A2/metabolismo , Proteínas S100/metabolismo , Animales , Anexina A2/química , Humanos , Conformación Proteica , Proteínas S100/químicaRESUMEN
Recent target validation studies have shown that inhibition of the protein interaction between annexin A2 and the S100A10 protein may have potential therapeutic benefits in cancer. Virtual screening identified certain 3,4,5-trisubstituted 4H-1,2,4-triazoles as moderately potent inhibitors of this interaction. A series of analogues were synthesized based on the 1,2,4-triazole scaffold and were evaluated for inhibition of the annexin A2-S100A10 protein interaction in competitive binding assays. 2-[(5-{[(4,6-Dimethylpyrimidin-2-yl)sulfanyl]methyl}-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)sulfanyl]-N-[4-(propan-2-yl)phenyl]acetamide (36) showed improved potency and was shown to disrupt the native complex between annexin A2 and S100A10.
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Anexina A2/antagonistas & inhibidores , Diseño de Fármacos , Triazoles/farmacología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/químicaRESUMEN
INTRODUCTION: Breast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice. METHODS: More than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer 'stem' cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account. RESULTS: The 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working. CONCLUSIONS: With resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.
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Neoplasias de la Mama , Investigación , Investigación Biomédica Traslacional , Animales , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/epidemiología , Neoplasias de la Mama/etiología , Neoplasias de la Mama/terapia , Femenino , HumanosRESUMEN
Annexin A2 (AnxA2) and S100A10 are known to form a molecular complex. Using fluorescence-based binding assays, we show that both proteins are localised on the cell surface, in a molecular form that allows mutual interaction. We hypothesized that binding between these proteins could facilitate cell-cell interactions. For cells that express surface S100A10 and surface annexin A2, cell-cell interactions can be blocked by competing with the interaction between these proteins. Thus an annexin A2-S100A10 molecular bridge participates in cell-cell interactions, revealing a hitherto unexplored function of this protein interaction.
Asunto(s)
Anexina A2/metabolismo , Neoplasias de la Mama/patología , Adhesión Celular/fisiología , Endotelio Vascular/citología , Proteínas S100/metabolismo , Anexina A2/fisiología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Células Cultivadas , Femenino , Humanos , Unión Proteica , Proteínas S100/fisiologíaRESUMEN
Protein interactions are increasingly appreciated as targets in small-molecule drug discovery. The interaction between the adapter protein S100A10 and its binding partner annexinâ A2 is a potentially important drug target. To obtain small-molecule starting points for inhibitors of this interaction, a three-dimensional pharmacophore model was constructed from the X-ray crystal structure of the complex between S100A10 and annexin A2. The pharmacophore model represents the favourable hydrophobic and hydrogen bond interactions between the two partners, as well as spatial and receptor site constraints (excluded volume spheres). Using this pharmacophore model, UNITY flex searches were carried out on a 3D library of 0.7 million commercially available compounds. This resulted in 568 hit compounds. Subsequently, GOLD docking studies were performed on these hits, and a set of 190 compounds were purchased and tested biochemically for inhibition of the protein interaction. Three compounds of similar chemical structure were identified as genuine inhibitors of the binding of annexin A2 to S100A10. The binding modes predicted by GOLD were in good agreement with their UNITY-generated conformations. We synthesised a series of analogues revealing areas critical for binding. Thus computational predictions and biochemical screening can be used successfully to derive novel chemical classes of protein-protein interaction blockers.
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Anexina A2/metabolismo , Diseño de Fármacos , Proteínas S100/metabolismo , Triazoles/química , Anexina A2/antagonistas & inhibidores , Sitios de Unión , Simulación por Computador , Bases de Datos de Compuestos Químicos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Mapas de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas S100/antagonistas & inhibidores , Programas Informáticos , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/metabolismoRESUMEN
S100 proteins are small adaptors that regulate the activity of partner proteins by virtue of direct protein interactions. Here, we describe the first small molecule blockers of the interaction between S100A10 and annexin A2. Molecular docking yielded candidate blockers that were screened for competition of the binding of an annexin A2 peptide to S100A10. Several inhibitory clusters were identified with some containing compounds with potency in the lower micromolar range. We chose 3-hydroxy-1-(2-hydroxypropyl)-5-(4-isopropylphenyl)-4-(4-methylbenzoyl)-1H-pyrrol-2(5H)-one (1a) as a starting point for structure-activity studies. These confirmed the hypothetical binding mode from the virtual screen for this series of molecules. Selected compounds disrupted the physiological complex of annexin A2 and S100A10, both in a broken cell preparation and inside MDA-MB-231 breast cancer cells. Thus, this class of compounds has promising properties as inhibitors of the interaction between annexin A2 and S100A10 and may help to elucidate the cellular function of this protein interaction.
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Anexina A2/metabolismo , Diseño de Fármacos , Pirroles/química , Pirroles/farmacología , Proteínas S100/metabolismo , Anexina A2/química , Evaluación Preclínica de Medicamentos , Ligandos , Modelos Moleculares , Fragmentos de Péptidos/metabolismo , Unión Proteica/efectos de los fármacos , Conformación Proteica , Pirroles/síntesis química , Proteínas S100/química , Relación Estructura-Actividad , Interfaz Usuario-ComputadorRESUMEN
Cytosolic components of the NADPH oxidase interact with the actin cytoskeleton. These interactions are thought to be important for the activation of this enzyme system but they are poorly characterised at the molecular level. Here we have explored the interaction between the actin cytoskeleton and p40(phox), one of the cytosolic components of NADPH oxidase. Full length p40(phox) expressed in COS cells co-localised with F-actin in a peripheral lamellar compartment. The co-localisation was lost after deletion of the Phox homology (PX) domain and the PX domain in isolation (p40PX) showed the same F-actin co-localisation as the full length protein. PX domains are known lipid-binding modules however, a mutant p40PX which did not bind lipids still co-localised with F-actin suggesting that lipid-independent interactions underlie the localisation. Affinity chromatography identified actin as a binding partner for p40PX in neutrophil extracts. Pure actin interacted with both p40(phox) and with p40PX suggesting it is a direct interaction. Disruption of the actin cytoskeleton with cytochalasin D resulted in actin rearrangement and concomitantly the localisation of full length p40(phox) proteins and that of p40PX changed. Thus p40PX is a dual F-actin/lipid-binding module and F-actin interactions with the PX domain dictate at least in part the intracellular localisation of the cytosolic p40(phox) subunit of the NADPH oxidase.
Asunto(s)
Actinas/metabolismo , NADPH Oxidasas/metabolismo , Neutrófilos/metabolismo , Fosfoproteínas/metabolismo , Transporte de Proteínas , Actinas/antagonistas & inhibidores , Animales , Células COS , Extractos Celulares , Chlorocebus aethiops , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Humanos , Neutrófilos/efectos de los fármacos , Neutrófilos/patología , Fosfoproteínas/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Dominios y Motivos de Interacción de Proteínas/genética , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Eliminación de Secuencia/genéticaRESUMEN
Protein-protein interactions are increasingly of interest as targets in small-molecule drug discovery. The interaction between the Ca2+- and phospholipid-binding protein Annexin A2 and its binding partner S100A10 has been implicated in angiogenesis and cancer metastasis. Here, we present a methodology to screen for inhibitors of this protein interaction. We developed a Cy5-labeled S100A10 tracer and showed by circular dichroism spectroscopy that the secondary structure is indistinguishable from that of non-labeled S100A10. This tracer was used to develop a binding assay based upon fluorescence resonance energy transfer to a Cy3-labeled Annexin A2 peptide ligand. The binding parameters matched those for unlabeled components as observed by equilibrium dialysis, which we determined separately, as well as those determined by isothermal titration calorimetry. Binding of labeled and unlabeled peptide was specific and mutually competitive. We used this assay for screening a small compound library derived by computational interrogation of the S100A10-binding pocket. Hits were obtained with IC(50) values in range of the IC(50) of the cognate Annexin A2 peptide ligand. Hits were subjected to an exact parallel assay measuring an unrelated protein-protein interaction (antigen-antibody). In this way, we identified genuine hits that inhibited the interaction between S100A10 and Annexin A2 but do not affect the fluorescence readout. These compounds are potentially of interest as candidates for further analysis and medical chemistry exploration. The simple assay format described here can be employed in early-stage exploration of other protein-protein interaction targets.
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Anexina A2/antagonistas & inhibidores , Anexina A2/metabolismo , Carbocianinas , Evaluación Preclínica de Medicamentos/métodos , Proteínas S100/metabolismo , Secuencia de Aminoácidos , Transferencia Resonante de Energía de Fluorescencia , Datos de Secuencia MolecularRESUMEN
Members of the protein kinase C family are major effectors of lipid second messengers. We describe three protocols to assess protein kinase C activity in polymorphonuclear leukocytes (neutrophils). These methods are useful to study the activation and function of protein kinase C in these immune cells. Since neutrophils provide a ready source of human primary tissue, these methods are also useful for pharmacological studies on the protein kinase C system and for evaluation of protein kinase C activators and inhibitors in the context of human primary cells. Furthermore, since protein kinase C activity is determined by a number of lipid-generating signaling systems, the methods described here can also be employed to study the pharmacology of these "upstream" signaling systems.
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Metabolismo de los Lípidos , Proteína Quinasa C/análisis , Proteína Quinasa C/metabolismo , Sistemas de Mensajero Secundario , Citosol/metabolismo , Activación Enzimática , Humanos , Espacio Intracelular/metabolismo , Isoenzimas/análisis , Isoenzimas/metabolismo , Microdominios de Membrana/metabolismo , NADPH Oxidasas , Neutrófilos/citología , Neutrófilos/enzimologíaAsunto(s)
Bioensayo/métodos , Sistemas de Liberación de Medicamentos/métodos , Diseño de Fármacos , Perfilación de la Expresión Génica/métodos , Preparaciones Farmacéuticas/administración & dosificación , Proteoma/metabolismo , Transducción de Señal/fisiología , Transducción de Señal/efectos de los fármacosRESUMEN
The tetrodotoxin-resistant voltage-gated sodium channel alpha-subunit Nav1.8 is expressed in nociceptors and has been implicated in chronic pain. Difficulties of heterologous expression have so far precluded analysis of the pharmacological properties of human Nav1.8. To address this we have introduced human Nav1.8 in neuroblastoma SH-SY5Y cells. Voltage-clamp analysis showed that human Nav1.8 generated an inward tetrodotoxin-resistant sodium current with an activating threshold around -50 mV, half maximal activation at -11+/-3 mV and a reversal potential of 67+/-4 mV. These properties closely match those of the endogenous rat tetrodotoxin-resistant sodium current in dorsal root ganglia suggesting that the expressed human channel is in a near physiological conformation. Human Nav1.8 was resistant to tetrodotoxin and activated by the pyrethroid toxin deltamethrin. Both voltage-activated and deltamethrin-activated human Nav1.8 were inhibited by the sodium channel blockers BIII 890 CL, NW-1029, and mexiletine. Inhibition of Nav1.8 by these compounds may underlie their known analgesic effects in animal models.
Asunto(s)
ARN Mensajero/metabolismo , Canales de Sodio/metabolismo , Amidas/farmacología , Animales , Anexina A2/genética , Anexina A2/metabolismo , Benzomorfanos/farmacología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Concentración 50 Inhibidora , Potenciales de la Membrana/efectos de los fármacos , Mexiletine/farmacología , Canal de Sodio Activado por Voltaje NAV1.8 , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma , Nitrilos/farmacología , Propionatos/farmacología , Piretrinas/farmacología , Ratas , Proteínas S100/genética , Proteínas S100/metabolismo , Sodio/metabolismo , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/efectos de los fármacos , Canales de Sodio/genética , Tetrodotoxina/farmacología , TransfecciónRESUMEN
G protein-gated inward rectifier (Kir3) channels are inhibited by activation of G(q/11)-coupled receptors and this has been postulated to involve the signaling molecules protein kinase C (PKC) and/or phosphatidylinositol 4,5-bisphosphate (PIP(2)). Their precise roles in mediating the inhibition of this family of channels remain controversial. We examine here their relative roles in causing inhibition of Kir3.1/3.2 channels stably expressed in human embryonic kidney (HEK)-293 cells after muscarinic M(3) receptor activation. In perforated patch mode, staurosporine prevented the G(q/11)-mediated, M(3) receptor, inhibition of channel activity. Recovery from M(3)-mediated inhibition was wortmannin sensitive. Whole cell currents, where the patch pipette was supplemented with PIP(2), were still irreversibly inhibited by M(3) receptor stimulation. When adenosine A(1) receptors were co-expressed, inclusion of PIP(2) rescued the A(1)-mediated response. Recordings from inside-out patches showed that catalytically active PKC applied directly to the intracellular membrane face inhibited the channels: a reversible effect modulated by okadaic acid. Generation of mutant heteromeric channel Kir3.1S185A/Kir3.2C-S178A, still left the channel susceptible to receptor, pharmacological, and direct kinase-mediated inhibition. Biochemically, labeled phosphate is incorporated into the channel. We suggest that PKC-delta mediates channel inhibition because recombinant PKC-delta inhibited channel activity, M(3)-mediated inhibition of the channel, was counteracted by overexpression of two types of dominant negative PKC-delta constructs, and, by using confocal microscopy, we have demonstrated translocation of green fluorescent protein-tagged PKC-delta to the plasma membrane on M(3) receptor stimulation. Thus Kir3.1/3.2 channels are sensitive to changes in membrane phospholipid levels but this is contingent on the activity of PKC-delta after M(3) receptor activation in HEK-293 cells.
Asunto(s)
Canales de Potasio de Rectificación Interna/fisiología , Proteína Quinasa C/metabolismo , Receptor Cross-Talk/fisiología , Receptor Muscarínico M3/fisiología , Línea Celular , Membrana Celular , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Humanos , Riñón/citología , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosforilación , Proteína Quinasa C-delta , Proteína Quinasa C-epsilonRESUMEN
Osteopontin (OPN) is an important chemokinetic agent for several cell types. Our earlier studies have shown that its expression is essential for uridine triphosphate (UTP)-mediated migration of vascular smooth muscle cells. We demonstrated previously that the activation of an AP-1 binding site located 76 bp upstream of the transcription start in the rat OPN promoter is involved in the induction of OPN expression. In this work, using a luciferase promoter deletion assay, we identified a new region of the rat OPN promoter (-1837 to -1757) that is responsive to UTP. This region contains an NFkappaB site located at -1800 and an Ebox located at -1768. Supershift electrophoretic mobility shift assay and chromatin immunoprecipitation assays identified NFkappaB and USF-1/USF-2 as the DNA binding proteins induced by UTP, respectively, for these two sites. Using dominant negative mutants of IkappaB kinase and USF transcription factors, we confirmed that NFkappaB and USF-1/USF-2 are involved in the UTP-mediated expression of OPN. Using a pharmacological approach, we demonstrated that USF proteins are regulated by the extracellular signal-regulated kinase (ERK)1/2 pathway, just as the earlier discovered AP-1 complex, whereas NFkappaB is up-regulated through PKCdelta signals. Finally, our work suggests that the UTP-stimulated OPN expression involves a coordinate regulation of PKCdelta-NFkappaB, ERK1/2-USF, and ERK1/2/NAD(P)H oxidase AP-1 signaling pathways.