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2.
Int J Radiat Biol ; 96(6): 836-843, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32052678

RESUMO

Purpose: The present study was conducted to re-evaluate the effect of low-level 1800 MHz RF signals on RAS/MAPK activation in live cells.Material and methods: Using Bioluminescence Resonance Energy Transfer technique (BRET), we assessed the effect of Continuous wave (CW) and Global System for Mobile (GSM)-modulated 1800 MHz signals (up to 2 W/kg) on ERK and RAS kinases' activity in live HuH7 cells.Results: We found that radiofrequency field (RF) exposure for 24 h altered neither basal level of RAS and ERK activation nor the potency of phorbol-12-myristate-13-acetate (PMA) to activate RAS and ERK kinases. However, we found that exposure to GSM-modulated 1800 MHz signals at 2 W/kg decreased the PMA maximal efficacy to activate both RAS and ERK kinases' activity. Exposure with CW 1800 MHz signal at 2 W/kg only decreased maximal efficacy of PMA to activate ERK but not RAS. No effects of RF exposure at 0.5 W/kg was observed on maximal efficacy of PMA to activate either RAS or ERK whatever the signal used.Conclusions: Our results indicate that RF exposure decreases the efficiency of the cascade of events, which, from the binding of PMA to its receptor(s), leads to the activation of RAS and ERK kinases.

3.
Int J Radiat Biol ; 96(3): 411-418, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31746658

RESUMO

Aim: The Pasche research group has reported that tumor-specific electromagnetic field frequencies have physiological and potential anti-tumor effects in cells, animals, and humans. Our aim was to investigate whether these fields have similar effects on physiological parameters in murine tumor models.Methods: Human HuH7 or HEPG2 cells were implanted in the right flank of 8-week-old female RAG gamma 2 C immunodeficient mice. An oximeter was used to record systolic blood pressure (pulse) in free-roaming conscious mice. Mice pulses were recorded and analyzed using a in-house software that also controlled the low-frequency generator for modulating the 27.12 MHz carrier wave at selected frequencies.Results: We performed exposures using both systematic scans at low frequencies and at the pre-determined frequencies reported by the Pasche group as altering both pulse and tumor growth in humans. Those exposures produced no detectable change in physiological parameters of tumor-bearing mice.Conclusion: No tumor-related frequencies were found, neither using systematic scans of frequencies nor published specific frequencies. There might obviously be differences between animal and human models, but our approach did not confirm the physiological data of the human Pasche group data.

4.
Radiat Res ; 189(1): 95-103, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29059001

RESUMO

The existence of effects of radiofrequency field exposure at environmental levels on living tissues and organisms remains controversial, in particular regarding potential "nonthermal" effects produced in the absence of temperature elevation. Therefore, we investigated whether TRPV1, one of the most studied thermosensitive channels, can be activated by the heat produced by radiofrequency fields and by some specific nonthermal interaction with the fields. We have recently shown that TRPV1 activation can be assessed in real-time on live cells using the bioluminescence resonance energy transfer technique. Taking advantage of this innovative assay, we monitored TRPV1 thermal and chemical modes of activation under radiofrequency exposure at 1800 MHz using different signals (CW, GSM, UMTS, LTE, Wi-Fi and WiMAX) at specific absorption rates between 8 and 32 W/kg. We showed that, as expected, TRPV1 channels were activated by the heat produced by radiofrequency field exposure of transiently-transfected HEK293T cells, but found no evidence of TRPV1 activation in the absence of temperature elevation under radiofrequency field exposure. There was no evidence either that, at fixed temperature, radiofrequency exposure altered the maximal efficacy of the agonist Capsaicin to activate TRPV1.


Assuntos
Ondas de Rádio/efeitos adversos , Canais de Cátion TRPV/metabolismo , Termorreceptores/metabolismo , Termorreceptores/efeitos da radiação , Calmodulina/metabolismo , Capsaicina/farmacologia , Células HEK293 , Humanos , Termorreceptores/efeitos dos fármacos
5.
Biophys J ; 112(1): 87-98, 2017 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-28076819

RESUMO

Multiplexed bioluminescence resonance energy transfer (BRET) assays were developed to monitor the activation of several functional transient receptor potential (TRP) channels in live cells and in real time. We probed both TRPV1 intramolecular rearrangements and its interaction with Calmodulin (CaM) under activation by chemical agonists and temperature. Our BRET study also confirmed that: (1) capsaicin and heat promoted distinct transitions, independently coupled to channel gating, and that (2) TRPV1 and Ca2+-bound CaM but not Ca2+-free CaM were preassociated in resting live cells, while capsaicin activation induced both the formation of more TRPV1/CaM complexes and conformational changes. The BRET assay, based on the interaction with Calmodulin, was successfully extended to TRPV3 and TRPV4 channels. We therefore developed a full-spectral three-color BRET assay for analyzing the specific activation of each of the three TRPV channels in a single sample. Such key improvement in BRET measurement paves the way for the simultaneous monitoring of independent biological pathways in live cells.


Assuntos
Transferência de Energia , Medições Luminescentes , Canais de Cátion TRPV/química , Canais de Cátion TRPV/metabolismo , Técnicas Biossensoriais , Calmodulina/metabolismo , Células HEK293 , Temperatura Alta , Humanos
6.
IEEE Trans Biomed Eng ; 63(11): 2317-2325, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-26886964

RESUMO

In this paper, the dosimetric characterization of an EMF exposure setup compatible with real-time impedance measurements of adherent biological cells is proposed. The EMF are directly delivered to the 16-well format plate used by the commercial xCELLigence apparatus. Experiments and numerical simulations were carried out for the dosimetric analysis. The reflection coefficient was less than -10 dB up to 180 MHz and this exposure system can be matched at higher frequencies up to 900 and 1800 MHz. The specific absorption rate (SAR) distribution within the wells containing the biological medium was calculated by numerical finite-difference time domain simulations and results were verified by temperature measurements at 13.56 MHz. Numerical SAR values were obtained at the microelectrode level where the biological cells were exposed to EMF including 13.56, 900, and 1800 MHz. At 13.56 MHz, the SAR values, within the cell layer and the 270-µL volume of medium, are 1.9e3 and 3.5 W/kg/incident mW, respectively.


Assuntos
Simulação por Computador , Impedância Elétrica , Modelos Biológicos , Radiometria/instrumentação , Radiometria/métodos , Desenho de Equipamento
7.
Bioelectromagnetics ; 36(4): 287-93, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25846808

RESUMO

The present study focused on gap junctional intercellular communication (GJIC) as a target for biological effects of extremely low-frequency (ELF) magnetic field (MF) exposure. Fluorescence recovery after photobleaching microscopy (FRAP) was used to visualize diffusion of a fluorescent dye between NIH3T3 fibroblasts through gap junctions. The direct effect of 24 h exposure to 50 Hz MF at 0.4 or 1 mT on GJIC function was assessed in one series of experiments. The potential synergism of MF with an inhibitor of GJIC, phorbol ester (TPA), was studied in another series by observing FRAP when NIH3T3 cells were incubated with TPA for 1 h following 24 h exposure to MF. In contrast to other reports of ELF-MF effects on GJIC, under our experimental conditions we observed neither direct inhibition of GJIC nor synergism with TPA-induced inhibition from 50 Hz MF exposures.


Assuntos
Comunicação Celular , Junções Comunicantes , Campos Magnéticos , Animais , Corantes Fluorescentes/metabolismo , Cinética , Camundongos , Células NIH 3T3
8.
FASEB J ; 28(10): 4509-23, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25053617

RESUMO

G-protein-coupled receptors have been shown to assemble at least as dimers early in the biosynthetic path, but some evidence suggests that they can also form larger oligomeric complexes. Using the human chemokine receptors CXCR4 and CCR2 as models, we directly probed the existence of higher order homo- and heterooligomers in human embryonic kidney cells. Combining bimolecular fluorescence and luminescence complementation (BiFC, BiLC) with bioluminescence resonance energy transfer (BRET) assays, we show that CXCR4 and CCR2 can assemble as homo- and heterooligomers, forming at least tetramers. Selective activation of CCR2 with the human monocyte chemotactic protein 1 (MCP-1) resulted in trans-conformational rearrangement of the CXCR4 dimer with an EC50 of 19.9 nM, compatible with a CCR2 action. Moreover, MCP-1 promoted the engagement of Gαi1, Gα13, Gαz, and ßarrestin2 to the heterooligomer, resulting in calcium signaling that was synergistically potentiated on coactivation of CCR2 and CXCR4, demonstrating that complexes larger than dimers reach the cell surface as functional units. A mutation of CXCR4 (N119K), which prevents Gi activation, also affects the CCR2-promoted engagement of Gαi1 and ßarrestin2 by the heterooligomer, supporting the occurrence of transprotomer regulation. Together, the results demonstrate that homo- and heteromultimeric CXCR4 and CCR2 can form functional signaling complexes that have unique properties.


Assuntos
Arrestinas/metabolismo , Quimiocina CCL2/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Receptores CCR2/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais , Células HEK293 , Humanos , Ligação Proteica , Multimerização Proteica , Receptores CXCR4/genética , beta-Arrestinas
9.
Traffic ; 15(4): 383-400, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24405750

RESUMO

The molecular mechanisms regulating G protein-coupled receptors (GPCRs) trafficking from their site of synthesis in the endoplasmic reticulum (ER) to their site of function (the cell surface) remain poorly characterized. Using a bioluminescence resonance energy transfer-based proteomic screen, we identified a novel GPCR-interacting protein; the human cornichon homologue 4 (CNIH4). This previously uncharacterized protein is localized in the early secretory pathway where it interacts with members of the 3 family of GPCRs. Both overexpression and knockdown expression of CNIH4 caused the intracellular retention of GPCRs, indicating that this ER-resident protein plays an important role in GPCR export. Overexpression of CNIH4 at low levels rescued the maturation and cell surface expression of an intracellularly retained mutant form of the ß2-adrenergic receptor, further demonstrating a positive role of CNIH4 in GPCR trafficking. Taken with the co-immunoprecipitation of CNIH4 with Sec23 and Sec24, components of the COPII coat complex responsible for ER export, these data suggest that CNIH4 acts as a cargo-sorting receptor, recruiting GPCRs into COPII vesicles.


Assuntos
Retículo Endoplasmático/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Células COS , Chlorocebus aethiops , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/genética
10.
Reprod Toxicol ; 36: 1-5, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23178895

RESUMO

In recent decades, concern has been growing about decreasing fecundity and fertility in the human population. Exposure to non-ionizing electromagnetic fields (EMF), especially radiofrequency (RF) fields used in wireless communications has been suggested as a potential risk factor. For the first time, we evaluated the effects of exposure to the 2450MHz Wi-Fi signal (1h/day, 6days/week) on the reproductive system of male and female Wistar rats, pre-exposed to Wi-Fi during sexual maturation. Exposure lasted 3 weeks (males) or 2 weeks (females), then animals were mated and couples exposed for 3 more weeks. On the day before delivery, the fetuses were observed for lethality, abnormalities, and clinical signs. In our experiment, no deleterious effects of Wi-Fi exposure on rat male and female reproductive organs and fertility were observed for 1h per days. No macroscopic abnormalities in fetuses were noted, even at the critical level of 4W/kg.


Assuntos
Desenvolvimento Embrionário/efeitos da radiação , Desenvolvimento Fetal/efeitos da radiação , Infertilidade Feminina/etiologia , Infertilidade Masculina/etiologia , Ondas de Rádio/efeitos adversos , Maturidade Sexual/efeitos da radiação , Tecnologia sem Fio , Animais , Relação Dose-Resposta à Radiação , Implantação do Embrião/efeitos da radiação , Perda do Embrião/etiologia , Ingestão de Energia/efeitos da radiação , Feminino , Genitália Masculina/crescimento & desenvolvimento , Genitália Masculina/imunologia , Genitália Masculina/efeitos da radiação , Masculino , Exposição Materna/efeitos adversos , Tamanho do Órgão/efeitos da radiação , Ovário/crescimento & desenvolvimento , Ovário/imunologia , Ovário/efeitos da radiação , Exposição Paterna/efeitos adversos , Distribuição Aleatória , Ratos , Ratos Wistar
11.
Prog Biophys Mol Biol ; 107(3): 369-73, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21914452

RESUMO

Animal studies can contribute to addressing the issue of possible greater health risk for children exposed to 50-60 Hz extremely low frequency (ELF) magnetic fields (MFs), mostly in terms of teratological effects and cancer. Teratology has been extensively studied in animals exposed to ELF MFs but experiments have not established adverse developmental effects. Childhood leukaemia has been the only cancer consistently reported in epidemiological studies as associated with exposure to ELF MFs. This association has been the basis for the classification as "possibly carcinogenic to humans" by the International Agency for Research on Cancer in 2002. Animal experiments have provided only limited support for these epidemiological findings. However, none but one study used an animal model for acute lymphoblastic leukaemia (ALL), the main form of childhood leukaemia, and exposures to ELF MFs were not carried out over the whole pregnancy period, when the first hit of ALL is assumed to occur. Moreover, there are no generally accepted biophysical mechanisms that could explain carcinogenic effects of low-level MFs. The radical pair mechanism and related cryptochromes (CRY) molecules have recently been identified in birds and other non-mammalian species, as a sensor of the geomagnetic field, involved in navigation. The hypothesis has to be tested in mammalian models. CRY, which is part of the molecular circadian clock machinery, is a ubiquitous protein likely to be involved in cancer cell growth and DNA repair. In summary, we now have some clues to test for a better characterization of the interaction between ALL and ELF MFs exposure.


Assuntos
Campos Magnéticos/efeitos adversos , Modelos Animais , Animais , Bioensaio , Radicais Livres/metabolismo , Humanos , Neoplasias/etiologia , Teratologia
12.
J Biol Chem ; 284(24): 16595-608, 2009 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-19380586

RESUMO

Promyelocytic leukemia protein (PML) is a tumor suppressor acting as the organizer of subnuclear structures called PML nuclear bodies (NBs). Both covalent modification of PML by the small ubiquitin-like modifier (SUMO) and non-covalent binding of SUMO to the PML SUMO binding domain (SBD) are necessary for PML NB formation and maturation. PML sumoylation and proteasome-dependent degradation induced by the E3 ubiquitin ligase, RNF4, are enhanced by the acute promyelocytic leukemia therapeutic agent, arsenic trioxide (As2O3). Here, we established a novel bioluminescence resonance energy transfer (BRET) assay to dissect and monitor PML/SUMO interactions dynamically in living cells upon addition of therapeutic agents. Using this sensitive and quantitative SUMO BRET assay that distinguishes PML sumoylation from SBD-mediated PML/SUMO non-covalent interactions, we probed the respective roles of covalent and non-covalent PML/SUMO interactions in PML degradation and interaction with RNF4. We found that, although dispensable for As2O3-enhanced PML sumoylation and RNF4 interaction, PML SBD core sequence was required for As2O3- and RNF4-induced PML degradation. As confirmed with a phosphomimetic mutant, phosphorylation of a stretch of serine residues, contained within PML SBD was needed for PML interaction with SUMO-modified protein partners and thus for NB maturation. However, mutation of these serine residues did not impair As2O3- and RNF4-induced PML degradation, contrasting with the known role of these phosphoserine residues for casein kinase 2-promoted PML degradation. Altogether, these data suggest a model whereby sumoylation- and SBD-dependent PML oligomerization within NBs is sufficient for RNF4-mediated PML degradation and does not require the phosphorylation-dependent association of PML with other sumoylated partners.


Assuntos
Leucemia Promielocítica Aguda/metabolismo , Proteínas Nucleares/metabolismo , Proteína SUMO-1/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Antineoplásicos/farmacologia , Trióxido de Arsênio , Arsenicais/farmacologia , Proteínas de Bactérias/genética , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Técnicas In Vitro , Rim/citologia , Leucemia Promielocítica Aguda/patologia , Luciferases de Renilla/genética , Medições Luminescentes , Proteínas Luminescentes/genética , Mutagênese Sítio-Dirigida , Proteínas Nucleares/química , Proteínas Nucleares/genética , Óxidos/farmacologia , Proteína da Leucemia Promielocítica , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Proteína SUMO-1/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transfecção , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética
13.
J Biol Chem ; 283(36): 24659-72, 2008 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-18566454

RESUMO

Determining the role of lipid raft nanodomains in G protein-coupled receptor signaling remains fraught by the lack of assays directly monitoring rafts in native membranes. We thus combined extensive biochemical and pharmacological approaches to a nanoscale strategy based on bioluminescence resonance energy transfer (BRET) to assess the spatial and functional influence of cholesterol-rich liquid-ordered lipid nanodomains on beta2 adrenergic receptor (beta2AR) signaling. The data revealed that whereas beta2AR did not partition within liquid-ordered lipid phase, a pool of G protein and adenylyl cyclase (AC) were sequestered in these domains. Destabilization of the liquid-ordered phase by cholesterol depletion led to a lateral redistribution of Galphas and AC that favored interactions between the receptor and its signaling partners as assessed by BRET. This resulted in an increased basal and agonist-promoted beta2AR-stimulated cAMP production that was partially dampened as a result of constitutive protein kinase A-dependent phosphorylation and desensitization of the receptor. This restraining influence of nanodomains on beta2AR signaling was further substantiated by showing that liquid-ordered lipid phase stabilization using caveolin overexpression or increasing membrane cholesterol amount led to an inhibition of beta2AR-associated signaling. Given the emerging concept that clustering of receptors and effectors into signaling platforms contributes to the efficacy and selectivity of signal transduction, our results support a model whereby cholesterol-promoted liquid-ordered lipid phase-embedding Gs and AC allows their lateral separation from the receptor, thus restraining the basal activity and controlling responsiveness of beta2AR signaling machinery within larger signaling platforms.


Assuntos
Colesterol/metabolismo , Microdomínios da Membrana/metabolismo , Modelos Biológicos , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais/fisiologia , Adenilil Ciclases/metabolismo , Agonistas Adrenérgicos/farmacologia , Caveolinas/biossíntese , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
14.
J Biol Chem ; 282(8): 5111-5, 2007 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-17197449

RESUMO

Ligand binding to G protein-coupled receptors (GPCRs) is thought to induce changes in receptor conformation that translate into activation of downstream effectors. The link between receptor conformation and activity is still insufficiently understood, as current models of GPCR activation fail to take an increasing amount of experimental data into account. To elucidate structure-function relationships in GPCR activation, we used bioluminescence resonance energy transfer to directly assess the conformation of mutants of the chemokine receptor CXCR4. We analyzed substitutions in the arginine cage DRY motif and in the conserved asparagine N(3.35)119, which are pivotal molecular switches for receptor conformation and activation. G(alpha)(i) activation of the mutants was either similar to wild-type CXCR4 (D133N, Y135A, and N119D) or resulted in loss of activity (R134A and N119K). Mutant N119S was constitutively active but further activated by agonist. Bioluminescence resonance energy transfer analysis suggested no simple correlation between conformational changes in response to ligand binding and activation of G(alpha)(i) by the mutants. Different conformations of active receptors were detected (for wild-type CXCR4, D133N, and N119S), suggesting that different receptor conformations are able to trigger G(alpha)(i) activity. Several conformations were also found for inactive mutants. These data provide biophysical evidence for different receptor conformations being active with respect to a single readout. They support models of GPCR structure-activity relationships that take this conformational flexibility of active receptors into account.


Assuntos
Substituição de Aminoácidos , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Modelos Biológicos , Receptores CXCR4/metabolismo , Animais , Linhagem Celular , Humanos , Conformação Proteica , Receptores CXCR4/genética , Transdução de Sinais/genética
15.
Nat Struct Mol Biol ; 13(9): 778-86, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16906158

RESUMO

Activation of heterotrimeric G proteins by their cognate seven transmembrane domain receptors is believed to involve conformational changes propagated from the receptor to the G proteins. However, the nature of these changes remains unknown. We monitored the conformational rearrangements at the interfaces between receptors and G proteins and between G protein subunits by measuring bioluminescence resonance energy transfer between probes inserted at multiple sites in receptor-G protein complexes. Using the data obtained for the alpha(2A)AR-G alpha(i1) beta1gamma2 complex and the available crystal structures of G alpha(i1) beta1gamma2, we propose a model wherein agonist binding induces conformational reorganization of a preexisting receptor-G protein complex, leading the G alpha-G betagamma interface to open but not dissociate. This conformational change may represent the movement required to allow nucleotide exit from the G alpha subunit, thus reflecting the initial activation event.


Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Sobrevivência Celular , Células Cultivadas , Transferência de Energia , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Espectrometria de Fluorescência
16.
J Virol ; 80(17): 8582-92, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16912307

RESUMO

PML nuclear bodies (NBs) are dynamic intranuclear structures harboring numerous transiently or permanently localized proteins. PML, the NBs' organizer, is directly induced by interferon, and its expression is critical for antiviral host defense. We describe herein the molecular events following poliovirus infection that lead to PML-dependent p53 activation and protection against virus infection. Poliovirus infection induces PML phosphorylation through the extracellular signal-regulated kinase pathway, increases PML SUMOylation, and induces its transfer from the nucleoplasm to the nuclear matrix. These events result in the recruitment of p53 to PML NBs, p53 phosphorylation on Ser15, and activation of p53 target genes leading to the induction of apoptosis. Moreover, the knock-down of p53 by small interfering RNA results in higher poliovirus replication, suggesting that p53 participates in antiviral defense. This effect, which requires the presence of PML, is transient since poliovirus targets p53 by inducing its degradation in a proteasome- and MDM2-dependent manner. Our results provide evidence of how poliovirus counteracts p53 antiviral activity by regulating PML and NBs, thus leading to p53 degradation.


Assuntos
Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Poliovirus/patogenicidade , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Apoptose , Linhagem Celular Tumoral , Estruturas do Núcleo Celular/metabolismo , Humanos , Organelas/metabolismo , Fosforilação , Proteína da Leucemia Promielocítica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteína Supressora de Tumor p53/genética , Replicação Viral
17.
Curr Protoc Neurosci ; Chapter 5: Unit 5.23, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18428639

RESUMO

Bioluminescence resonance energy transfer (BRET) allows monitoring of protein-protein interactions in real time in living cells. One candidate interacting protein is fused to a luminescent energy donor, such as Renilla luciferase, and the other to a fluorescent energy acceptor, such the green fluorescent protein (GFP), and the two are then coexpressed in the same cells. If the two proteins interact, their close proximity allows nonradiative energy transfer (BRET) between the luciferase and the GFP. BRET does not occur if the two proteins are separated by more than 100 A, making the technique ideal for monitoring protein-protein interactions in biological systems. This unit describes the use of BRET to study constitutive and agonist-promoted interactions among signaling molecules, as illustrated by the homodimerization of the CXCR4 receptor and the recruitment of beta-arrestin2 to agonist-activated G-protein-coupled receptors. This noninvasive and homogeneous assay provides a robust and sensitive proteomic platform with applications for basic science research and drug discovery.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Proteínas/metabolismo , Animais , Linhagem Celular Transformada , Expressão Gênica/fisiologia , Humanos , Proteínas Luminescentes/genética , Microscopia de Fluorescência/instrumentação , Proteínas/química , Proteínas/genética , Transfecção/métodos
18.
Mol Pharmacol ; 67(6): 1966-76, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15761117

RESUMO

CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor that governs migration of leukocytes and serves as a coreceptor for the R5 tropic strains of human immunodeficiency virus (HIV). CCR5-mediated signaling in response to CC chemokines relies on G protein activation. Desensitization, which rapidly turns off G protein-dependent signaling, involves phosphorylation of CCR5 that promotes interaction of the receptor with beta-arrestins for endocytosis. Whether coupling to G proteins, desensitization, and endocytosis of CCR5 require the same structural determinants remains a matter of investigation. Here, we show that CCR5 displayed agonist-independent coupling to G proteins. This constitutive activity of the receptor was abrogated by TAK779 (N,N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]carbonyl]amino]benzyl]tetrahydro-2H-pyran-4-aminium chloride), a nonpeptidic CCR5 ligand that inhibits HIV infection and was found to depend on the integrity of the Asp-Arg-Tyr (DRY) motif. Changing Arg-126 by the neutral residue Asn (R126N-CCR5 mutant) abolished CCR5-mediated activation of G proteins, either constitutively or in response to agonists. In contrast, R126N-CCR5 not only retained agonist-promoted phosphorylation and beta-arrestin-dependent endocytosis but also displayed a higher basal phosphorylation than wild-type CCR5. Expression of beta-arrestin in R126N-CCR5-expressing cells resulted in receptor down-regulation, thereby suggesting that R126N-CCR5 spontaneously interacts with beta-arrestins. However, although expression of beta-arrestin favored wild-type CCR5-mediated chemotaxis, it failed to promote migration of cells expressing R126N-CCR5. Overall, these data indicate that structural requirements for CCR5-mediated activation of G proteins, albeit not involved in receptor desensitization and internalization, are needed for beta-arrestin-mediated chemotaxis. These results have implications for how distinct biological responses of CCR5 might rely on a different set of receptor conformations.


Assuntos
Endocitose/fisiologia , Mutação , Receptores CCR5/genética , Receptores CCR5/fisiologia , Transdução de Sinais/fisiologia , Motivos de Aminoácidos , Animais , Arrestinas/metabolismo , Arrestinas/farmacologia , Linhagem Celular , Cricetinae , Relação Dose-Resposta a Droga , Endocitose/efeitos dos fármacos , Humanos , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Receptores CCR5/química , Transdução de Sinais/efeitos dos fármacos , beta-Arrestinas
19.
J Biol Chem ; 280(11): 9895-903, 2005 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-15632118

RESUMO

Homo- and heterodimerization have emerged as prominent features of G-protein-coupled receptors with possible impact on the regulation of their activity. Using a sensitive bioluminescence resonance energy transfer system, we investigated the formation of CXCR4 and CCR2 chemokine receptor dimers. We found that both receptors exist as constitutive homo- and heterodimers and that ligands induce conformational changes within the pre-formed dimers without promoting receptor dimer formation or disassembly. Ligands with different intrinsic efficacies yielded distinct bioluminescence resonance energy transfer modulations, indicating the stabilization of distinct receptor conformations. We also found that peptides derived from the transmembrane domains of CXCR4 inhibited activation of this receptor by blocking the ligand-induced conformational transitions of the dimer. Taken together, our data support a model in which chemokine receptor homo- and heterodimers form spontaneously and respond to ligand binding as units that undergo conformational changes involving both protomers even when only one of the two ligand binding sites is occupied.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Receptores CXCR4/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Linhagem Celular , Quimiocina CCL2/metabolismo , AMP Cíclico/metabolismo , Dimerização , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Cinética , Ligantes , Proteínas Luminescentes/metabolismo , Peptídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Temperatura , Fatores de Tempo , Transfecção
20.
J Biol Chem ; 279(30): 31398-408, 2004 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-15133044

RESUMO

The HIV-1 Nef protein is a critical virulence factor that exerts multiple effects during viral replication. Nef modulates surface expression of various cellular proteins including CD4 and MHC-I, enhances viral infectivity, and affects signal transduction pathways. Nef has been shown to partially associate with rafts, where it can prime T cells for activation. The contribution of rafts during Nef-induced CD4 down-regulation and enhancement of viral replication remains poorly understood. We show here that Nef does not modify the palmitoylation state of CD4 or its partition within rafts. Moreover, CD4 mutants lacking palmitoylation or unable to associate with rafts are efficiently down-regulated by Nef. In HIV-infected cells, viral assembly and budding occurs from rafts, and Nef has been suggested to increase this process. However, using T cells acutely infected with wild-type or nef-deleted HIV, we did not observe any impact of Nef on raft segregation of viral structural proteins. We have also designed a palmitoylated mutant of Nef (NefG3C), which significantly accumulates in rafts. Interestingly, the efficiency of NefG3C to down-regulate CD4 and MHC-I, and to promote viral replication was not increased when compared with the wild-type protein. Altogether, these results strongly suggest that rafts are not a key element involved in the effects of Nef on trafficking of cellular proteins and on viral replication.


Assuntos
Antígenos CD4/metabolismo , Produtos do Gene nef/fisiologia , HIV-1/patogenicidade , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Células Cultivadas , Deleção de Genes , Genes nef , HIV-1/genética , HIV-1/fisiologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Células Jurkat , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/virologia , Mutação , Ácido Palmítico/metabolismo , Virulência , Produtos do Gene nef do Vírus da Imunodeficiência Humana
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