RESUMEN
Viruses manipulate the central machineries of host cells to their advantage. They prevent host cell antiviral responses to create a favorable environment for their survival and propagation. Measles virus (MV) encodes two nonstructural proteins MV-V and MV-C known to counteract the host interferon response and to regulate cell death pathways. Several molecular mechanisms underlining MV-V regulation of innate immunity and cell death pathways have been proposed, whereas MV-C host-interacting proteins are less studied. We suggest that some cellular factors that are controlled by MV-C protein during viral replication could be components of innate immunity and the cell death pathways. To determine which host factors are targeted by MV-C, we captured both direct and indirect host-interacting proteins of MV-C protein. For this, we used a strategy based on recombinant viruses expressing tagged viral proteins followed by affinity purification and a bottom-up mass spectrometry analysis. From the list of host proteins specifically interacting with MV-C protein in different cell lines, we selected the host targets that belong to immunity and cell death pathways for further validation. Direct protein interaction partners of MV-C were determined by applying protein complementation assay and the bioluminescence resonance energy transfer approach. As a result, we found that MV-C protein specifically interacts with p65-iASPP protein complex that controls both cell death and innate immunity pathways and evaluated the significance of these host factors on virus replication.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Represoras/metabolismo , Factor de Transcripción ReIA/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Muerte Celular , Línea Celular , Chlorocebus aethiops , Interacciones Huésped-Patógeno , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Virus del Sarampión/genética , Virus del Sarampión/fisiología , Mapas de Interacción de Proteínas , Proteómica , Proteínas Represoras/genética , Factor de Transcripción ReIA/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas no Estructurales Virales/genética , Replicación ViralRESUMEN
At the crossroad between the NF-κB and the MAPK pathways, the ternary complex composed of p105, ABIN2 and TPL2 is essential for the host cell response to pathogens. The matrix protein (M) of field isolates of rabies virus was previously shown to disturb the signaling induced by RelAp43, a NF-κB protein close to RelA/p65. Here, we investigated how the M protein disturbs the NF-κB pathway in a RelAp43-dependant manner and the potential involvement of the ternary complex in this mechanism. Using a tandem affinity purification coupled with mass spectrometry approach, we show that RelAp43 interacts with the p105-ABIN2-TPL2 complex and we observe a strong perturbation of this complex in presence of M protein. M protein interaction with RelAp43 is associated with a wide disturbance of NF-κB signaling, involving a modulation of IκBα-, IκBß-, and IκBε-RelAp43 interaction and a favored interaction of RelAp43 with the non-canonical pathway (RelB and p100/p52). Monitoring the interactions between host and viral proteins using protein-fragment complementation assay and bioluminescent resonance energy transfer, we further show that RelAp43 is associated to the p105-ABIN2-TPL2 complex as RelAp43-p105 interaction stabilizes the formation of a complex with ABIN2 and TPL2. Interestingly, the M protein interacts not only with RelAp43 but also with TPL2 and ABIN2. Upon interaction with this complex, M protein promotes the release of ABIN2, which ultimately favors the production of RelAp43-p50 NF-κB dimers. The use of recombinant rabies viruses further indicates that this mechanism leads to the control of IFNß, TNF and CXCL2 expression during the infection and a high pathogenicity profile in rabies virus infected mice. All together, our results demonstrate the important role of RelAp43 and M protein in the regulation of NF-κB signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas , Complejos Multiproteicos/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Virus de la Rabia/metabolismo , Rabia/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Células HEK293 , Células HeLa , Humanos , Quinasas Quinasa Quinasa PAM/genética , Complejos Multiproteicos/genética , Subunidad p50 de NF-kappa B/genética , Proteínas Proto-Oncogénicas/genética , Rabia/genética , Virus de la Rabia/genética , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Fluorescence and bioluminescence resonance energy transfer (FRET, BRET) techniques are powerful tools for studying protein-protein interactions in cellular assays. In contrast to fluorescent proteins, chemiluminescent proteins do not require excitation light, known to trigger autofluorescence, phototoxicity, and photobleaching. Regrettably, low signal intensity of luciferase systems restricts their usage as they require specialized microscopes equipped with ultra low-light imaging cameras. In this study, we report that bioluminescence quantification in living cells using a standard widefield automated microscope dedicated to screening and high content analysis is possible with the newer luciferase systems, Nanoluciferase (Nluc). With such equipment, we showed that robust intramolecular BRET can be measured using a combination of Nluc and yellow fluorescent protein (YFP). Using the human Superoxide Dismutase 1 (SOD1) dimer model, we next validated that intermolecular BRET could be quantified at a single cell level. The enhanced signal brightness of Nluc enabling BRET imaging to widefield microscopy shows strong potential to open up single cell protein-protein interactions studies to a wider audience. © 2016 International Society for Advancement of Cytometry.
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Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Nanotecnología , Proteínas Bacterianas/química , Línea Celular , Humanos , Luciferasas/química , Proteínas Luminiscentes/química , Microscopía , Mapas de Interacción de Proteínas/genética , Superóxido Dismutasa-1/químicaRESUMEN
More than 100 copper/zinc superoxide dismutase 1 (SOD1) genetic mutations have been characterized. These mutations lead to the death of motor neurons in ALS. In its native form, the SOD1 protein is expressed as a homodimer in the cytosol. In vitro studies have shown that SOD1 mutations impair the dimerization kinetics of the protein, and in vivo studies have shown that SOD1 forms aggregates in patients with familial forms of ALS. In this study, we analyzed WT SOD1 and 9 mutant (mt) forms of the protein by non-invasive fluorescence techniques. Using microscopic techniques such as fluorescence resonance energy transfer, fluorescence complementation, image-based quantification, and fluorescence correlation spectroscopy, we studied SOD1 dimerization, oligomerization, and aggregation. Our results indicate that SOD1 mutations lead to an impairment in SOD1 dimerization and, subsequently, affect protein aggregation. We also show that SOD1 WT and mt proteins can dimerize. However, aggregates are predominantly composed of SOD1 mt proteins.
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Esclerosis Amiotrófica Lateral/enzimología , Hígado/enzimología , Mutación , Multimerización de Proteína , Superóxido Dismutasa/química , Superóxido Dismutasa/genética , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Hígado/citología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Estructura Cuaternaria de Proteína , Espectrometría de Fluorescencia , Superóxido Dismutasa-1RESUMEN
Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and PARK2/Parkin mutations cause autosomal recessive forms of Parkinson's disease. Upon a loss of mitochondrial membrane potential (DeltaPsi(m)) in human cells, cytosolic Parkin has been reported to be recruited to mitochondria, which is followed by a stimulation of mitochondrial autophagy. Here, we show that the relocation of Parkin to mitochondria induced by a collapse of DeltaPsi(m) relies on PINK1 expression and that overexpression of WT but not of mutated PINK1 causes Parkin translocation to mitochondria, even in cells with normal DeltaPsi(m). We also show that once at the mitochondria, Parkin is in close proximity to PINK1, but we find no evidence that Parkin catalyzes PINK1 ubiquitination or that PINK1 phosphorylates Parkin. However, co-overexpression of Parkin and PINK1 collapses the normal tubular mitochondrial network into mitochondrial aggregates and/or large perinuclear clusters, many of which are surrounded by autophagic vacuoles. Our results suggest that Parkin, together with PINK1, modulates mitochondrial trafficking, especially to the perinuclear region, a subcellular area associated with autophagy. Thus by impairing this process, mutations in either Parkin or PINK1 may alter mitochondrial turnover which, in turn, may cause the accumulation of defective mitochondria and, ultimately, neurodegeneration in Parkinson's disease.
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Autofagia/fisiología , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/metabolismo , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Carbonil Cianuro m-Clorofenil Hidrazona/metabolismo , Línea Celular , Humanos , Ionóforos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Mitocondrias/ultraestructura , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Unión Proteica , Proteínas Quinasas/genética , Transporte de Proteínas/fisiología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
We have developed a novel fluorogenic nanoprobe prepared from the assembly of CdSe/ZnS quantum dot (QD) and gold (Au) nanoparticles in which QD was conjugated with a specifically designed ß-secretase (BACE1) substrate peptide, which was allowed to bind to the Ni-nitrilotriacetate (Ni-NTA) modified Au nanoparticles. This coordination-mediated binding of the QD with Au nanoparticles via Ni-NTA-histidine (His) interaction resulted in highly efficient quenching of QD fluorescence through a distance-dependent fluorescence resonance energy transfer (FRET) phenomenon. The prequenched QD-Au assembly recovered the fluorescence in the presence of the BACE1 enzyme after incubation in vitro. The high quenching efficiency of AuNP and robust QD fluorescence signal recovery upon BACE1 enzymatic digestion enabled us to visualize BACE1 activity in living cells, which further allowed us to generate the half maximal inhibitory concentration (IC(50)) values for BACE1 inhibitors in the cell-based assay utilizing a high throughput system (HTS). These results suggest the potential application of QD-AuNP assembly toward the HTS drug screening system as a robust and efficient probe to identify active molecules in BACE1-related diseases such as Alzheimer's disease.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Transferencia Resonante de Energía de Fluorescencia/métodos , Oro/química , Nanopartículas/química , Puntos Cuánticos , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/química , Colorantes Fluorescentes/química , Células HEK293 , Humanos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismoRESUMEN
Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful method to visualize and quantify protein-protein interaction in living cells. Unfortunately, the emission bleed-through of FPs limits the usage of this complex technique. To circumvent undesirable excitation of the acceptor fluorophore, using two-photon excitation, we searched for FRET pairs that show selective excitation of the donor but not of the acceptor fluorescent molecule. We found this property in the fluorescent cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) and YFP/mCherry FRET pairs and performed two-photon excited FRET spectral imaging to quantify protein interactions on the later pair that shows better spectral discrimination. Applying non-negative matrix factorization to unmix two-photon excited spectral imaging data, we were able to eliminate the donor bleed-through as well as the autofluorescence. As a result, we achieved FRET quantification by means of a single spectral acquisition, making the FRET approach not only easy and straightforward but also less prone to calculation artifacts. As an application of our approach, the intermolecular interaction of amyloid precursor protein and the adaptor protein Fe65 associated with Alzheimer's disease was quantified. We believe that the FRET approach using two-photon and fluorescent YFP/mCherry pair is a promising method to monitor protein interaction in living cells.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Luminiscentes/metabolismo , Fotones , Mapeo de Interacción de Proteínas/métodos , Algoritmos , Proteínas Bacterianas/metabolismo , Supervivencia Celular , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Unión ProteicaRESUMEN
Despite the adaptation of major clinical imaging modalities for small animals, optical bioluminescence imaging technology is the main approach readily reporting gene activity. Yet, in vivo bioluminescence monitoring requires the administration and diffusion of a substrate to the tissues of interest, resulting in experimental variability, high reagent cost, long acquisition time, and stress to the animal. In our study, we avoid such issues upon generating a new transgenic mouse (GFAP-E2crimson) expressing the far-red fluorescent protein E2-crimson under the control of the glial fibrillary acidic protein (GFAP) promoter. Using microscopy, we validated the selective expression of the reporter in the astrocyte cell population and by non-invasive in vivo fluorescence imaging its detection through the scalps and skulls of live animals. In addition, we performed a longitudinal study validating by in vivo imaging that the E2-crimson fluorescence signal is up-regulated, in pups during astrogenesis and in adult mice during astrogliosis upon kainic acid administration. Furthermore, upon crossing GFAP-E2crimson transgenic with 5XFAD Alzheimer's disease mice model, we were able to quantify the chronic inflammation triggered by amyloid deposit and aging over 18 months. As many diseases and conditions can trigger neuroinflammation, we believe that the GFAP-E2crimson reporter mice model delivers tremendous value for the non-invasive quantification of astrogliosis responses in living animals.
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Gliosis , Ácido Kaínico , Animales , Astrocitos/metabolismo , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Proteína Ácida Fibrilar de la Glía/metabolismo , Gliosis/metabolismo , Estudios Longitudinales , Ratones , Ratones TransgénicosRESUMEN
Luminometer and imaging systems are used to detect and quantify low light produced by a broad range of bioluminescent proteins. Despite their everyday use in research, such instruments are costly and lack the flexibility to accommodate the variety of bioluminescence experiment formats that may require top or bottom signal acquisition, high or medium sensitivity, or multiple wavelength detection. To address the growing need for versatile technologies, we developed a highly customizable bioluminescence imager called Biolum' RGB that uses a consumer color digital camera with a high-aperture lens mounted at the bottom or top of a 3D-printed dark chamber and can quantify bioluminescence emission from cells grown in 384-well microplates and Petri dishes. Taking advantage of RGB detectors, Biolum' RGB can distinguish spectral signatures from various bioluminescence probes and quantify bioluminescence resonant energy transfer occurring during protein-protein interaction events. Although Biolum' RGB can be used with any smartphone, in particular for low bioluminescence signals, we recommend the use of recent digital cameras which offer better sensitivity and high signal/noise ratio. Altogether, Biolum' RGB combines the benefits of a plate reader and imager while providing better image resolution and faster acquisition speed, and as such, it offers an exciting alternative for any laboratory looking for a versatile, low-cost bioluminescence imaging instrument.
Asunto(s)
Diagnóstico por Imagen , Teléfono Inteligente , Proteínas Luminiscentes/metabolismoRESUMEN
Bioluminescence resonance energy transfer (BRET) saturation is a method of studying protein-protein interaction (PPI) upon quantification of the dependence of the BRET signal on the acceptor/donor (A:D) expression ratio. In this study, using the very bright Nluc/YFP BRET pair acquired respectively with microplate reader and automated confocal microscopy, we significantly improved BRET saturation assay by extending A:D expression detection range and normalizing A:D expression with a new BRET-free probe. We next found that upon using variable instead of fixed amount of donor molecules co-expressed with increasing acceptor concentrations, BRET saturation assay robustness can be further improved when studying cytosolic protein, although the relative amounts of dimers (BRETmax) and the relative dimer affinity (BRET50) remain similar. Altogether, we show that our method can be applied to many PPI networks, involving the NF-κB pathway, high-affinity nanobody, rabies virus-host interactions, mTOR complex and JAK/STAT signaling. Altogether our approach paves the way for robust PPI validation and characterization in living cells.
Asunto(s)
Mediciones Luminiscentes , Mapas de Interacción de Proteínas , Bioensayo , Transferencia de Energía , Mediciones Luminiscentes/métodos , Transducción de SeñalRESUMEN
Combining high throughput screening approaches with induced pluripotent stem cell (iPSC)-based disease modeling represents a promising unbiased strategy to identify therapies for neurodegenerative disorders. Here we applied high content imaging on iPSC-derived neurons from patients with familial Parkinson's disease bearing the G209A (p.A53T) α-synuclein (αSyn) mutation and launched a screening campaign on a small kinase inhibitor library. We thus identified the multi-kinase inhibitor BX795 that at a single dose effectively restores disease-associated neurodegenerative phenotypes. Proteomics profiling mapped the molecular pathways underlying the protective effects of BX795, comprising a cohort of 118 protein-mediators of the core biological processes of RNA metabolism, protein synthesis, modification and clearance, and stress response, all linked to the mTORC1 signaling hub. In agreement, expression of human p.A53T-αSyn in neuronal cells affected key components of the mTORC1 pathway resulting in aberrant protein synthesis that was restored in the presence of BX795 with concurrent facilitation of autophagy. Taken together, we have identified a promising small molecule with neuroprotective actions as candidate therapeutic for PD and other protein conformational disorders.
RESUMEN
Rabies is a zoonotic disease caused by rabies virus (RABV). As rabies advances, patients develop a variety of severe neurological symptoms that inevitably lead to coma and death. Unlike other neurotropic viruses that can induce symptoms of a similar range, RABV-infected post-mortem brains do not show significant signs of inflammation nor the structural damages on neurons. This suggests that the observed neurological symptoms possibly originate from dysfunctions of neurons. However, many aspects of neuronal dysfunctions in the context of RABV infection are only partially understood, and therefore require further investigation. In this study, we used differentiated neurons to characterize the RABV-induced transcriptomic changes at the early time-points of infection. We found that the genes modulated in response to the infection are particularly involved in cell cycle, gene expression, immune response, and neuronal function-associated processes. Comparing a wild-type RABV to a mutant virus harboring altered matrix proteins, we found that the RABV matrix protein plays an important role in the early down-regulation of host genes, of which a significant number is involved in neuronal functions. The kinetics of differentially expressed genes (DEGs) are also different between the wild type and mutant virus datasets. The number of modulated genes remained constant upon wild-type RABV infection up to 24 h post-infection, but dramatically increased in the mutant condition. This result suggests that the intact viral matrix protein is important to control the size of host gene modulation. We then examined the signaling pathways previously studied in relation to the innate immune responses against RABV, and found that these pathways contribute to the changes in neuronal function-associated processes. We further examined a set of regulated genes that could impact neuronal functions collectively, and demonstrated in calcium imaging that indeed the spontaneous activity of neurons is influenced by RABV infection. Overall, our findings suggest that neuronal function-associated genes are modulated by RABV early on, potentially through the viral matrix protein-interacting signaling molecules and their downstream pathways.
RESUMEN
We introduce a fast spectral imaging system using an electron-multiplying charge-coupled device (EM-CCD) as a detector. Our system is combined with a custom-built two-photon excitation laser scanning microscope and has 80 detection channels, which allow for high spectral resolution and fast frame acquisition without any loss of spectral information. To demonstrate the efficiency of our approach, we applied this technology to monitor fluorescent proteins and quantum dot-labeled G protein-coupled receptors in living cells as well as autofluorescence in tissue samples.
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Aumento de la Imagen/instrumentación , Microscopía Confocal/instrumentación , Microscopía de Fluorescencia por Excitación Multifotónica/instrumentación , Espectrometría de Fluorescencia/instrumentación , Diseño de Equipo , Análisis de Falla de EquipoRESUMEN
The generation of compact quantum dots (QDs) probes is of critical importance for visualizing molecular interaction occurring in biological context, particularly when using the Förster resonance energy transfer (FRET) approach. This Article reports novel water-soluble compact CdSe/ZnS QDs prepared by ligand exchange reaction using thiolated nitrilotriacetate (NTA). The resulting NTA-QDs revealed higher stability and remarkable conjugation efficiency compared to the other QDs prepared with different ligands by using the ligand exchange method. The Ni-NTA group is a well-known binding moiety for the detection and purification of oligohistidine-tagged recombinant proteins. We demonstrated that NiNTA-QDs prepared by Ni(2+) complexation exhibited highly specific binding ability toward 6-histidine (His)-tagged peptides present in various experimental conditions (buffer solution, agarose beads, and HEK cells). Importantly, the compact NiNTA-QDs serve as an efficient FRET donor. These results suggest that the stable and highly selective multifunctional NTA-QDs can be useful for labeling and tracking molecular interactions within biological context.
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Transferencia Resonante de Energía de Fluorescencia , Ácido Nitrilotriacético/análogos & derivados , Compuestos Organometálicos/química , Péptidos/química , Puntos Cuánticos , Compuestos de Cadmio/química , Línea Celular , Histidina/química , Humanos , Ligandos , Ácido Nitrilotriacético/química , Proteínas Recombinantes/química , Compuestos de Selenio/química , Solubilidad , Sulfuros/química , Agua/química , Compuestos de Zinc/químicaRESUMEN
We have synthesized 50 benzimidazole (BMZ) derivatives with 1,2-phenylenediamines and aromatic aldehydes under mild oxidation conditions by using inexpensive, nontoxic inorganic salt sodium metabisulfite in a one-pot condensation reaction and screened their ability to interfere with Zika virus (ZIKV) infection utilizing a cell-based phenotypic assay. Seven BMZs inhibited an African ZIKV strain with a selectivity index (SI=CC50 /EC50 ) of 9-37. Structure-activity relationship analysis demonstrated that substitution at the C-2, N-1, and C-5 positions of the BMZ ring were important for anti-ZIKV activity. The hybrid structure of BMZ and naphthalene rings was a structural feature responsible for the high anti-ZIKV activity. Importantly, BMZs inhibited ZIKV in human neural stem cells, a physiologically relevant system considering the severe congenital anomalies, like microcephaly, caused by ZIKV infection. Compound 39 displayed the highest antiviral efficacy against the African ZIKV strain in Huh-7 (SI>37) and neural stem cells (SI=12). Compound 35 possessed the highest activity in Vero cells (SI=115). Together, our data indicate that BMZs derivatives have to be considered for the development of ZIKV therapeutic interventions.
Asunto(s)
Antivirales/farmacología , Bencimidazoles/farmacología , Virus Zika/efectos de los fármacos , Aldehídos/química , Aldehídos/farmacología , Antivirales/síntesis química , Antivirales/química , Bencimidazoles/síntesis química , Bencimidazoles/química , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/virología , Fenilendiaminas/química , Fenilendiaminas/farmacología , Relación Estructura-ActividadRESUMEN
Throughout the rabies virus (RABV) infectious cycle, host-virus interactions define its capacity to replicate, escape the immune response, and spread. As phosphorylation is a key regulatory mechanism involved in most cellular processes, kinases represent a target of choice to identify host factors required for viral replication. A kinase and phosphatase small interfering RNA (siRNA) high-content screening was performed on a fluorescent protein-recombinant field isolate (Tha RABV). We identified 57 high-confidence key host factors important for RABV replication with a readout set at 18 h postinfection and 73 with a readout set at 36 h postinfection, including 24 common factors at all stages of the infection. Amongst them, gene clusters of the most prominent pathways were determined. Up to 15 mitogen-activated protein kinases (MAPKs) and effectors, including MKK7 (associated with Jun N-terminal protein kinase [JNK] signalization) and DUSP5, as well as 17 phosphatidylinositol (PI)-related proteins, including PIP5K1C and MTM1, were found to be involved in the later stage of RABV infection. The importance of these pathways was further validated, as small molecules Ro 31-8820 and PD 198306 inhibited RABV replication in human neurons.IMPORTANCE Rabies virus relies on cellular machinery for its replication while simultaneously evading the host immune response. Despite their importance, little is known about the key host factors required for rabies virus infection. Here, we focused on the human kinome, at the core of many cellular pathways, to unveil a new understanding of the rabies virus infectious cycle and to discover new potential therapeutic targets in a small interfering RNA screening. The mitogen-activated protein kinase pathway and phosphatidylinositol metabolism were identified as prominent factors involved in rabies virus infection, and those findings were further confirmed in human neurons. While bringing a new insight into rabies virus biology, we also provide a new list of host factors involved in rabies virus infection.
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Interacciones Microbiota-Huesped , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositoles/metabolismo , Interferencia de ARN , Virus de la Rabia/fisiología , Línea Celular , Ensayos Analíticos de Alto Rendimiento , Humanos , Redes y Vías Metabólicas , Virus de la Rabia/genética , Bibliotecas de Moléculas Pequeñas , Replicación ViralRESUMEN
We have studied the fluorescence decays of the purified enhanced cyan fluorescent protein (ECFP, with chromophore sequence Thr-Trp-Gly) and of its variant carrying the single H148D mutation characteristic of the brighter form Cerulean. Both proteins exhibit highly complex fluorescence decays showing strong temperature and pH dependences. At neutral pH, the H148D mutation leads (i) to a general increase in all fluorescence lifetimes and (ii) to the disappearance of a subpopulation, estimated to be more than 25% of the total ECFP molecules, characterized by a quenched and red-shifted fluorescence. The fluorescence lifetime distributions of ECFP and its H148D mutant remain otherwise very similar, indicating a high degree of structural and dynamic similarity of the two proteins in their major form. From thermodynamic analysis, we conclude that the multiexponential decay of ECFP cannot be simply ascribed, as is generally admitted, to the slow conformational exchange characterized by NMR and X-ray crystallographic studies [Seifert, M. H., et al. (2002) J. Am. Chem. Soc. 124, 7932-7942; Bae, J. H., et al. (2003) J. Mol. Biol. 328, 1071-1081]. Parallel measurements in living cells show that these fluorescence properties in neutral solution are very similar to those of cytosolic ECFP.
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Fluorescencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación , Animales , Línea Celular , Proteínas Fluorescentes Verdes/química , Humanos , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Proteínas Mutantes/química , Conformación Proteica , Espectrometría de Fluorescencia , Temperatura , TermodinámicaRESUMEN
Nicotine consumed upon smoking affects numerous physiological processes through nicotinic acetylcholine receptors, which mediate cholinergic regulation by the neuronal and endogenous acetylcholine. Consequently, nicotinic receptors are expressed in many non-excitable tissues including the blood. In spite of the documented effect of nicotine on hematopoiesis, little is known about the expression and role of nicotinic receptors in the course of blood cell differentiation. The aim of the present study was to investigate whether and how nicotinic receptors are involved in the development of myeloid and erythroid cells within the bone marrow. The presence of nicotinic receptors containing alpha4(beta2) and alpha7 subunits in the bone marrow cells of C57Bl/6 mice was shown by the binding of [125I]-alpha-bungarotoxin or [3H]-Epibatidine and by flow cytometry with subunit-specific antibodies or fluorescein-labeled alpha-cobratoxin. Both TER119+ (erythroid) and CD16+CD43med (myeloid) progenitor cells bound more alpha4-specific antibodies than their mature forms, while the binding of alpha-cobratoxin and alpha7-specific antibodies was also high in mature cells. According to morphological analysis, either the absence of alpha7-containing nicotinic receptors in knockout mice or their desensitization in mice chronically treated with nicotine decreased the number of myeloid and erythroid progenitors and junior cells. In contrast, the absence of beta2-containing receptors favored myelocyte generation and erythroid cell maturation. It is concluded that the development of both myeloid and erythroid cell lineages is regulated by endogenous cholinergic ligands and can be affected by nicotine through alpha7- and alpha4beta2-containing nicotinic receptors, which play different roles in the course of the cell maturation.
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Eritropoyesis , Mielopoyesis , Receptores Nicotínicos/fisiología , Animales , Anticuerpos , Células de la Médula Ósea/química , Células de la Médula Ósea/citología , Citometría de Flujo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Nicotínicos/genética , Receptores Nicotínicos/inmunología , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Ni-nitrilotriacetic acid (NTA) functionalized CdSe/ZnS quantum dots (QDs) were exploited as a site-specific labeling agent of histidine-tagged biomolecules in live cells; the QDs were found to be water-soluble, aggregation free and stable for several months.
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Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Histidina/química , Níquel/química , Ácido Nitrilotriacético/química , Proteínas/química , Puntos Cuánticos , Coloración y Etiquetado/métodos , Línea Celular , Supervivencia Celular/efectos de los fármacos , HumanosRESUMEN
During aggressive tumor growth and migration, glioblastoma cells secrete diverse molecules and adhesion proteins to the extracellular matrix. Yet, the biochemical effects of the glioblastoma secretome in the brain remain largely unknown. Here we show that soluble CD44 secreted from glioblastoma cells induces neuronal degeneration through the activation of tau pathology in the brain. Glioblastoma-xenograft tissues showed a number of degenerating neurons bearing highly phosphorylated tau. Through a series of secretome-analyses, we identified that soluble CD44 was the responsible protein inducing tau phosphorylation and aggregation (EC50 = 19.1 ng/mL). The treatment of sCD44 to primary hippocampal neurons-induced tau hyperphosphorylation, leading to neuronal degeneration. Also, the injection of sCD44 into the brains of tau transgenic mice induced tau hyper-phosphorylation in hippocampal neurons. Altogether, our data suggest a neurodegenerative role of sCD44 in promoting tau pathology and serving as a molecular link between glioblastoma and neurodegeneration.