RESUMO
Gßγ subunits mediate many different signaling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear Gßγ signaling, we investigated the functional role of Gßγ signaling in the regulation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. We identified a novel, negative, regulatory role for the Gß1γ dimer in the fibrotic response. Depletion of Gß1 led to derepression of the fibrotic response at the mRNA and protein levels under basal conditions and an enhanced fibrotic response after sustained stimulation of the angiotensin II type I receptor. Our genome-wide chromatin immunoprecipitation experiments revealed that Gß1 colocalized and interacted with RNA polymerase II on fibrotic genes in an angiotensin II-dependent manner. Additionally, blocking transcription with inhibitors of Cdk9 prevented association of Gßγ with transcription complexes. Together, our findings suggest that Gß1γ is a novel transcriptional regulator of the fibrotic response that may act to restrict fibrosis to conditions of sustained fibrotic signaling. Our work expands the role for Gßγ signaling in cardiac fibrosis and may have broad implications for the role of nuclear Gßγ signaling in other cell types.
Assuntos
Fibroblastos , Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Regulação da Expressão Gênica , Miocárdio , RNA Polimerase II , Transcrição Gênica , Animais , Ratos , Angiotensina II/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Fibroblastos/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transdução de Sinais/fisiologia , Miocárdio/citologia , Miocárdio/patologia , FibroseRESUMO
BACKGROUND: Cryptococcal meningitis has fatality rates of 40%-70%, resulting in 200 000 deaths each year. The best outcomes are achieved with amphotericin combined with flucytosine but flucytosine is expensive and unavailable where most disease occurs. More effective and affordable treatments are needed. Tamoxifen, a selective oestrogen receptor modulator frequently indicated for breast cancer, has been found to have synergistic activity against the Cryptococcus neoformans type strain when combined with amphotericin or fluconazole. It is cheap, off-licence, widely available and well-tolerated, and thus a pragmatic potential treatment for cryptococcal disease. OBJECTIVES: We wanted to determine the susceptibility of clinical isolates of C. neoformans to tamoxifen alone and in combination with other antifungals, to determine whether there is sufficient evidence of activity to justify a clinical trial. METHODS: We used the CLSI broth microdilution protocol to test the susceptibility of 30 randomly selected clinical isolates of C. neoformans to tamoxifen, in dual combination with amphotericin, fluconazole or flucytosine, and in triple combination with amphotericin and fluconazole. Evidence of drug interactions was assessed using the fractional inhibitory concentration index. RESULTS: The MIC50 and MIC90 of tamoxifen were 4 and 16 mg/L, respectively. The combination of tamoxifen and amphotericin suggested a synergistic interaction in 20 of 30 (67%) isolates. There was no interaction between tamoxifen and either fluconazole or flucytosine. Synergy was maintained in 3-Dimensional chequerboard testing. There was no evidence of antagonism. CONCLUSIONS: Tamoxifen may be a useful addition to treatment with amphotericin and fluconazole for cryptococcal meningitis; a trial is justified.
Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Antagonistas de Estrogênios/farmacologia , Fluconazol/farmacologia , Tamoxifeno/farmacologia , Cryptococcus neoformans/isolamento & purificação , Sinergismo Farmacológico , Humanos , Meningite Criptocócica/tratamento farmacológico , Meningite Criptocócica/microbiologia , Testes de Sensibilidade MicrobianaRESUMO
At the cell surface, ßARs and endothelin receptors can regulate nitric oxide (NO) production. ß-adrenergic receptors (ßARs) and type B endothelin receptors (ETB) are present in cardiac nuclear membranes and regulate transcription. The present study investigated the role of the NO pathway in the regulation of gene transcription by these nuclear G protein-coupled receptors. Nitric oxide production and transcription initiation were measured in nuclei isolated from the adult rat heart. The cell-permeable fluorescent dye 4,5-diaminofluorescein diacetate (DAF2 DA) was used to provide a direct assessment of nitric oxide release. Both isoproterenol and endothelin increased NO production in isolated nuclei. Furthermore, a ß3AR-selective agonist, BRL 37344, increased NO synthesis whereas the ß1AR-selective agonist xamoterol did not. Isoproterenol increased, whereas ET-1 reduced, de novo transcription. The NO synthase inhibitor l-NAME prevented isoproterenol from increasing either NO production or de novo transcription. l-NAME also blocked ET-1-induced NO-production but did not alter the suppression of transcription initiation by ET-1. Inhibition of the cGMP-dependent protein kinase (PKG) using KT5823 also blocked the ability of isoproterenol to increase transcription initiation. Furthermore, immunoblotting revealed eNOS, but not nNOS, in isolated nuclei. Finally, caged, cell-permeable isoproterenol and endothelin-1 analogs were used to selectively activate intracellular ß-adrenergic and endothelin receptors in intact adult cardiomyocytes. Intracellular release of caged ET-1 or isoproterenol analogs increased NO production in intact adult cardiomyocytes. Hence, activation of the NO synthase/guanylyl cyclase/PKG pathway is necessary for nuclear ß3ARs to increase de novo transcription. Furthermore, we have demonstrated the potential utility of caged receptor ligands in selectively modulating signaling via endogenous intracellular G protein-coupled receptors.
Assuntos
Miócitos Cardíacos/metabolismo , Óxido Nítrico/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores de Endotelina/metabolismo , Animais , Endotelina-1/farmacologia , Isoproterenol/farmacologia , Masculino , Miócitos Cardíacos/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Quinolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Receptores Adrenérgicos beta/genética , Receptores de Endotelina/genética , Transdução de SinaisRESUMO
Background: Cryptococcal meningitis has high mortality. Flucytosine is a key treatment but is expensive and rarely available. The anticancer agent tamoxifen has synergistic anti-cryptococcal activity with amphotericin in vitro. It is off-patent, cheap, and widely available. We performed a trial to determine its therapeutic potential. Methods: Open label randomized controlled trial. Participants received standard care - amphotericin combined with fluconazole for the first 2 weeks - or standard care plus tamoxifen 300 mg/day. The primary end point was Early Fungicidal Activity (EFA) - the rate of yeast clearance from cerebrospinal fluid (CSF). Trial registration https://clinicaltrials.gov/ct2/show/NCT03112031. Results: Fifty patients were enrolled (median age 34 years, 35 male). Tamoxifen had no effect on EFA (-0.48log10 colony-forming units/mL/CSF control arm versus -0.49 tamoxifen arm, difference -0.005log10CFU/ml/day, 95% CI: -0.16, 0.15, p=0.95). Tamoxifen caused QTc prolongation. Conclusions: High-dose tamoxifen does not increase the clearance rate of Cryptococcus from CSF. Novel, affordable therapies are needed. Funding: The trial was funded through the Wellcome Trust Asia Programme Vietnam Core Grant 106680 and a Wellcome Trust Intermediate Fellowship to JND grant number WT097147MA.
Assuntos
Anfotericina B/uso terapêutico , Cryptococcus neoformans/efeitos dos fármacos , Fluconazol/uso terapêutico , Meningite Criptocócica/tratamento farmacológico , Tamoxifeno/uso terapêutico , Adulto , Antifúngicos/uso terapêutico , Quimioterapia Combinada , Feminino , Humanos , Síndrome do QT Longo/induzido quimicamente , Masculino , Meningite Criptocócica/líquido cefalorraquidiano , Meningite Criptocócica/metabolismo , Tamoxifeno/efeitos adversosRESUMO
Several therapeutic compounds have been identified that prolong the QT interval on the electrocardiogram and cause torsade de pointes arrhythmias not by direct block of the cardiac potassium channel human ether-à-go-go-related gene (hERG) but via disruption of hERG trafficking to the cell surface membrane. One example of a clinically important compound class that potently inhibits hERG trafficking are cardiac glycosides. We have shown previously that inhibition of hERG trafficking by cardiac glycosides is initiated via direct block of Na(+)/K(+) pumps and not via off-target interactions with hERG or any other protein. However, it was not known how pump inhibition at the cell surface is coupled to hERG processing in the endoplasmic reticulum. Here, we show that depletion of intracellular K(+)-either indirectly after long-term exposure to cardiac glycosides or directly after exposure to gramicidin in low sodium media-is sufficient to disrupt hERG trafficking. In K(+)-depleted cells, hERG trafficking can be restored by permeating K(+) or Rb(+) ions, incubation at low temperature, exposure to the pharmacological chaperone astemizole, or specific mutations in the selectivity filter of hERG. Our data suggest a novel mechanism for drug-induced trafficking inhibition in which cardiac glycosides produce a [K(+)](i)-mediated conformational defect directly in the hERG channel protein.
Assuntos
Retículo Endoplasmático/metabolismo , Canais de Potássio Éter-A-Go-Go/metabolismo , Líquido Intracelular/metabolismo , Potássio/metabolismo , Glicosídeos Cardíacos/farmacologia , Linhagem Celular , Relação Dose-Resposta a Droga , Retículo Endoplasmático/efeitos dos fármacos , Humanos , Líquido Intracelular/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologiaRESUMO
In the current model of gamma-aminobutyric acid (GABA) B receptor function, there is a requirement for GABA-B(1/2) heterodimerisation for targetting to the cell surface. However, different lines of evidence suggest that the GABA-B(1) subunit can form a functional receptor in the absence of GABA-B(2). We observed coupling of endogenous GABA-B(1) receptors in the DI-TNC1 glial cell line to the ERK pathway in response to baclofen even though these cells do not express GABA-B(2). GABA-B(1A) receptors were also able to mediate a rapid, transient, and dose-dependent activation of the ERK1/2 MAP kinase pathway when transfected alone into HEK 293 cells. The response was abolished by G(i/o) and MEK inhibition, potentiated by inhibitors of phospholipase C and protein kinase C and did not involve PI-3-kinase activity. Finally, using bioluminescence resonance energy transfer and co-immunoprecipitation, we show the existence of homodimeric GABA-B(1A) receptors in transfected HEK293 cells. Altogether, our observations show that GABA-B(1A) receptors are able to activate the ERK1/2 pathway despite the absence of surface targetting partner GABA-B(2) in both HEK 293 cells and the DI-TNC1 cell line.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptores de GABA-B/metabolismo , Animais , Baclofeno/farmacologia , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/química , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , GABAérgicos/farmacologia , Agonistas GABAérgicos/farmacologia , Agonistas dos Receptores de GABA-B , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/antagonistas & inibidores , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Toxina Pertussis/farmacologia , Fosforilação , Multimerização Proteica , Ratos , Ácido gama-Aminobutírico/farmacologiaRESUMO
Recent studies have demonstrated that adenylyl cyclase isoforms can form both homo- and heterodimers and that this may be the basic functional unit of these enzymes (see Cooper, D.M.F. and Crossthwaite, A.J. (2006) Trends. Pharmacol. Sci. 8:426-431). Here, we show that adenylyl cyclases 2 and 5 can form a functional heterodimeric complex in HEK293 cells using a combination of BRET, confocal imaging, co-immunoprecipitation and assays of adenylyl cyclase activity. The AC2/5 complex is formed constitutively and is stable in the presence of receptor or forskolin stimulation. The complex formed by AC2/5 is also much more sensitive to the presence of Galpha(s) and forskolin than either of the parent AC isoforms themselves. Finally, we also show that this complex can be detected in native tissues as AC2 and AC5 were localized to the same structures in adult mouse ventricular myocytes and neonatal mouse cardiac fibroblasts and could be co-immunoprecipitated from lysates of mouse heart.
Assuntos
Adenilil Ciclases/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Isoenzimas/metabolismo , Transdução de Sinais , Adenilil Ciclases/genética , Animais , Animais Recém-Nascidos , Linhagem Celular , Colforsina/farmacologia , AMP Cíclico/metabolismo , Dimerização , Relação Dose-Resposta a Droga , Ativação Enzimática , Ativadores de Enzimas/farmacologia , Transferência Ressonante de Energia de Fluorescência , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Imunoprecipitação , Isoenzimas/genética , Luciferases de Renilla/metabolismo , Camundongos , Microscopia Confocal , Norepinefrina/metabolismo , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismo , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 1/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
A variety of fluorescent proteins with different spectral properties have been created by mutating green fluorescent protein. When these proteins are split in two, neither fragment is fluorescent per se, nor can a fluorescent protein be reconstituted by co-expressing the complementary N- and C-terminal fragments. However, when these fragments are genetically fused to proteins that associate with each other in cellulo, the N- and C-terminal fragments of the fluorescent protein are brought together and can reconstitute a fluorescent protein. A similar protein complementation assay (PCA) can be performed with two complementary fragments of various luciferase isoforms. This makes these assays useful tools for detecting the association of two proteins in living cells. Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) occurs when energy from, respectively, a luminescent or fluorescent donor protein is non-radiatively transferred to a fluorescent acceptor protein. This transfer of energy can only occur if the proteins are within 100A of each other. Thus, BRET and FRET are also useful tools for detecting the association of two proteins in living cells. By combining different protein fragment complementation assays (PCA) with BRET or FRET it is possible to demonstrate that three or more proteins are simultaneous parts of the same protein complex in living cells. As an example of the utility of this approach, we show that as many as four different proteins are simultaneously associated as part of a G protein-coupled receptor signalling complex.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Complexos Multiproteicos/análise , Proteínas Recombinantes de Fusão/análise , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bioensaio/métodos , Linhagem Celular Transformada , Linhagem Celular Tumoral , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Luciferases/análise , Luciferases/genética , Luciferases/metabolismo , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Plasmídeos , Mapeamento de Interação de Proteínas/métodos , Multimerização Proteica , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/efeitos da radiação , Projetos de Pesquisa , Espectrometria de Fluorescência/métodos , TransfecçãoRESUMO
OBJECTIVE: We sought to determine if different beta-adrenergic receptor (betaAR) subtypes, and their associated signalling machinery, are functionally localized to nuclear membranes. METHODS: Employing enriched nuclear preparations, we assayed the specific presence of betaAR by measuring 125I-cyanopindolol (CYP) binding, Western blotting, confocal microscopy and functional assays. RESULTS: Western blots of rat heart nuclear fractions and confocal immunofluorescent analysis of adult rat and mouse ventricular cardiomyocytes displayed the presence of beta 1AR and beta 3AR but, surprisingly, not the beta 2AR on nuclear membranes. Nuclear localization of downstream signalling partners Gs, Gi and adenylyl cyclases II and V/VI was also demonstrated. The functional relevance of nuclear betaAR was shown by receptor-mediated stimulation of adenylyl cyclase activity by isoproterenol but not the beta 3AR-selective agonist CL 316243 in enriched nuclear preparations. We also examined the effect of subtype-selective ligands on the initiation of RNA synthesis in isolated nuclei. Both isoproterenol and another beta 3AR-selective agonist, BRL 37344, increased RNA synthesis which was inhibited by pertussis toxin (PTX). Neither a beta 1AR-selective agonist, xamoterol, nor a beta 2AR-selective agonist, procaterol, was able to stimulate transcription. However, both CGP 20712A and ICI 118,551 blocked isoproterenol-mediated effects to varying extents. PTX treatment also revealed that nuclear betaAR may be coupled to other signalling pathways in addition to Gi, as stimulation under these conditions reduced initiation of transcription below basal levels. CONCLUSION: These results highlight differential subcellular localization for betaAR subtypes and indicate that betaAR may have specific roles in regulating nuclear function in cardiomyocytes.
Assuntos
Miócitos Cardíacos/metabolismo , Membrana Nuclear/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais/fisiologia , Adenilil Ciclases/metabolismo , Agonistas de Receptores Adrenérgicos beta 3 , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Western Blotting/métodos , Dioxóis/farmacologia , Etanolaminas/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Ventrículos do Coração , Imidazóis/farmacologia , Iodocianopindolol/metabolismo , Isoproterenol/farmacologia , Camundongos , Microscopia Confocal/métodos , Membrana Nuclear/química , Toxina Pertussis/farmacologia , Propanolaminas/farmacologia , Ratos , Receptores Adrenérgicos beta 1/análise , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/análise , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 3/análise , Receptores Adrenérgicos beta 3/metabolismoRESUMO
GPCRs form signalling complexes with other receptors as part of dimers, G proteins and effector partners. A proteomic screen to identify proteins that associate with the ß2-adrenergic receptor (ß2AR) identified many of components of the Endoplasmic-Reticulum-Associated Degradation (ERAD) quality control system [1], including the valosin-containing protein (VCP/p97). Here, we validated the interaction of VCP with co-expressed FLAG-ß2AR, demonstrating, using an inducible expression system, that the interaction of FLAG-ß2AR and VCP is not an artifact of overexpression of the ß2AR per se. We knocked down VCP and noted that levels of FLAG-ß2AR were increased in cells with lower VCP levels. This increase in the level of FLAG-ß2AR did not lead to an increase in the level of functional receptor observed at the cell surface. Similarly, inhibition of the proteasome lead to a dramatic increase in the abundance of TAP-ß2AR, while cellular responses again remained unchanged. Taken together, our data suggests that a substantial proportion of the ß2AR produced is non-functional and VCP plays a key role in the maturation and trafficking of the ß2AR as part of the ERAD quality control process.
Assuntos
Biossíntese de Proteínas , Receptores Adrenérgicos beta 2/biossíntese , Proteína com Valosina/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Ligação Proteica/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacosRESUMO
The vast burden of cryptococcal meningitis occurs in immunosuppressed patients, driven by HIV, and is caused by Cryptococcus neoformans var. grubii. We previously reported cryptococcal meningitis in Vietnam arising atypically in HIV uninfected, apparently immunocompetent patients, caused by a single amplified fragment length polymorphism (AFLP) cluster of C. neoformans var. grubii (VNIγ). This variant was less common in HIV infected individuals; it remains unclear why this lineage is associated with apparently immunocompetent patients. To study this host tropism we aimed to further our understanding of clinical phenotype and genomic variation within Vietnamese C. neoformans var. grubii. After performing MLST on C. neoformans clinical isolates we identified 14 sequence types (STs); ST5 correlated with the VNIγ cluster. We next compared clinical phenotype by lineage and found HIV infected patients with cryptococcal meningitis caused by ST5 organisms were significantly more likely to have lymphadenopathy (11% vs. 4%, p = 0.05 Fisher's exact test) and higher blood lymphocyte count (median 0.76 versus 0.55 X109 cells/L, p = 0.001, Kruskal-Wallis test). Furthermore, survivors of ST5 infections had evidence of worse disability outcomes at 70 days (72.7% (40/55) in ST5 infections versus 57.1% (52/91) non-ST5 infections (OR 2.11, 95%CI 1.01 to 4.41), p = 0.046). To further investigate the relationship between strain and disease phenotype we performed genome sequencing on eight Vietnamese C. neoformans var. grubii. Eight genome assemblies exhibited >99% nucleotide sequence identity and we identified 165 kbp of lineage specific to Vietnamese isolates. ST5 genomes harbored several strain specific regions, incorporating 19 annotated coding sequences and eight hypothetical proteins. These regions included a phenolic acid decarboxylase, a DEAD-box ATP-dependent RNA helicase 26, oxoprolinases, a taurine catabolism dioxygenase, a zinc finger protein, membrane transport proteins and various drug transporters. Our work outlines the complexity of genomic pathogenicity in cryptococcal infections and identifies a number of gene candidates that may aid the disaggregation of the pathways associated with the pathogenesis of Cryptococcus neoformans var. grubii.
Assuntos
Cryptococcus neoformans/classificação , Cryptococcus neoformans/genética , Variação Genética , Genoma Fúngico , Infecções por HIV/complicações , Meningite Criptocócica/microbiologia , Cryptococcus neoformans/isolamento & purificação , Cryptococcus neoformans/fisiologia , Genômica , Especificidade de Hospedeiro , Humanos , Meningite Criptocócica/patologia , Tipagem de Sequências Multilocus , Técnicas de Tipagem Micológica , VietnãRESUMO
OBJECTIVE: To determine whether transient hypoxia in neonatal rats has long-lasting effects on inotropic stimulation of the adult heart. METHODS: The hearts of adult male Sprague-Dawley rats (89+/-1 (S.E.M.) days, 432+/-5 g) were studied. Half the animals had been subjected to neonatal hypoxia (FiO(2)=0.12, days 1-10) while the others had not. The peak response of left ventricular pressure (LVP) and the maximum rate of pressure increase (+dP/dtmax) were measured in isolated and perfused hearts during application of dobutamine, isoproterenol, milrinone and betaxolol. Left ventricular myocyte membranes were analyzed for beta receptor density, adenylyl cyclase activity and content. RESULTS: LVP and +dP/dtmax responses to stimulation with dobutamine and isoproterenol were significantly impaired in adult hearts of neonatally hypoxic rats. The inotropic effect of dobutamine was abolished by blockade with the beta(1)-selective antagonist betaxolol. The inotropic effects of the phosphodiesterase inhibitor milrinone were also significantly decreased in neonatally hypoxic adult hearts. There was no difference in left ventricular myocyte membrane beta receptor density of adult hearts whether they were hypoxic neonatally or not. However, left ventricular adenylyl cyclase activity on isoproterenol or forskolin stimulation and adenylyl cyclase levels (type V/VI) were significantly reduced in neonatally hypoxic adult hearts. CONCLUSIONS: Neonatal hypoxia in the rat has long-lasting effects on the left ventricular response to inotropic stimulation at maturity likely at least in part due to diminished left ventricular adenylyl cyclase levels.
Assuntos
Hipóxia/fisiopatologia , Função Ventricular Esquerda/efeitos dos fármacos , Antagonistas Adrenérgicos beta/farmacologia , Animais , Animais Recém-Nascidos , Peso Corporal , Cardiotônicos/farmacologia , Dobutamina/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Hipóxia/patologia , Isoproterenol/farmacologia , Masculino , Milrinona/farmacologia , Miocárdio/patologia , Tamanho do Órgão , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
Inositol pyrophosphates are cellular signals that are created by the actions of inositol kinases and are degraded by highly active inositol phosphatases. The potent actions of these phosphatases suggest these signals must be created near their sites of action. To identify sites where the inositol kinase, PPIP5K1 acts, we performed affinity purification of PPIP5K1 from HEK293 cells and analyzed these samples using mass spectrometry to identify the proteins pesent (10.1016/j.cellsig.2016.02.002) [1]. We further decreased PPIP5K1 levels in HeLa cells and treated these with PPIP5K1 siRNA. We then monitored the motility of these cells in Scratch assays.
RESUMO
Cellular signaling involves coordinated regulation of many events. Scaffolding proteins are crucial regulators of cellular signaling, because they are able to affect numerous events by coordinating specific interactions among multiple protein partners in the same pathway. Scaffolding proteins often contain intrinsically disordered regions (IDR) that facilitate the formation and function of distinct protein complexes. We show that PPIP5K1 contains an unusually long and evolutionarily conserved IDR. To investigate the biological role(s) of this domain, we identified interacting proteins using affinity purification coupled with mass spectrometry. Here, we report that PPIP5K1 is associated with a network of proteins that regulate vesicle-mediated transport. We further identified exocyst complex component 1 as a direct interactor with the IDR of PPIP5K1. Additionally, we report that knockdown of PPIP5K1 decreases motility of HeLa cells in a wound-healing assay. These results suggest that PPIP5K1 might play an important role in regulating function of exocyst complex in establishing cellular polarity and directional migration of cells.
Assuntos
Movimento Celular , Proteínas Intrinsicamente Desordenadas/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/química , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Transporte Biológico , Células HEK293 , Células HeLa , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/fisiologia , Espectrometria de Massas , Fosfotransferases (Aceptor do Grupo Fosfato)/fisiologia , Domínios Proteicos , Mapeamento de Interação de ProteínasRESUMO
Much is known about the how Gßγ subunits regulate effectors in response to G protein-coupled receptor stimulation. However, there is still a lot we don't know about how specific combinations of Gß and Gγ are wired into different signalling pathways. Here, using an siRNA screen for different Gß and Gγ subunits, we examined an endogenous M3 muscarinic receptor signalling pathway in HEK 293 cells. We observed that Gß(4) subunits were critical for calcium signalling and a downstream surrogate measured as ERK1/2 MAP kinase activity. A number of Gγ subunits could partner with Gß(4) but the best coupling was seen via Gß(4)γ(1). Intriguingly, knocking down Gß(1) actually increased signalling through the M3-mAChR most likely via an increase in Gß(4) levels. We noted that Gß(1) occupies the promoter of Gß(4) and may participate in maturation of its mRNA. This highlights a new role for Gßγ signalling beyond their canonical roles in cellular signalling.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Receptores Muscarínicos/metabolismo , Transdução de Sinais , Sítios de Ligação , Sinalização do Cálcio , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Relação Dose-Resposta a Droga , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Regiões Promotoras Genéticas , Multimerização Proteica , Interferência de RNA , RNA Mensageiro/metabolismo , Receptor Muscarínico M3 , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/genética , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica , TransfecçãoRESUMO
Opioid and α2-adrenoceptor (AR) agonists are analgesic when administered in the spinal cord and show a clinically beneficial synergistic interaction when co-administered. However, α2-AR antagonists can also inhibit opioid antinociception, suggesting a complex interaction between the two systems. The α2A-AR subtype is necessary for spinal adrenergic analgesia and synergy with opioids for most agonist combinations. Therefore, we investigated whether spinal opioid antinociception and opioid-adrenergic synergy were under allosteric control of the α2A-AR. Drugs were administered intrathecally in wild type (WT) and α2A-knock-out (KO) mice and antinociception was measured using the hot water tail immersion or substance P behavioral assays. The α2A-AR agonist clonidine was less effective in α2A-KO mice in both assays. The absence of the α2A-AR resulted in 10-70-fold increases in the antinociceptive potency of the opioid agonists morphine and DeltII. In contrast, neither morphine nor DeltII synergized with clonidine in α2A-KO mice, indicating that the α2AAR has both positive and negative modulatory effects on opioid antinociception. Depletion of descending adrenergic terminals with 6-OHDA resulted in a significant decrease in morphine efficacy in WT but not in α2A-KO mice, suggesting that endogenous norepinephrine acts through the α2A-AR to facilitate morphine antinociception. Based on these findings, we propose a model whereby ligand-occupied versus ligand-free α2A-AR produce distinct patterns of modulation of opioid receptor activation. In this model, agonist-occupied α2A-ARs potentiate opioid analgesia, while non-occupied α2A-ARs inhibit opioid analgesia. Exploiting such interactions between the two receptors could lead to the development of better pharmacological treatments for pain management.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Analgésicos Opioides/farmacologia , Dor Nociceptiva/tratamento farmacológico , Receptores Adrenérgicos alfa 2/metabolismo , Medula Espinal/efeitos dos fármacos , Regulação Alostérica , Animais , Clonidina/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Temperatura Alta , Injeções Espinhais , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dor Nociceptiva/metabolismo , Receptores Adrenérgicos alfa 2/genética , Medula Espinal/metabolismo , Substância PRESUMO
The maturation and folding of G protein-coupled receptors are governed by mechanisms that remain poorly understood. In an effort to characterize these biological events, we optimized a novel, gel-free proteomic approach to identify partners of the ß2-adrenergic receptor (ß2AR). In addition to a number of known interacting proteins such as heterotrimeric G protein subunits, this allowed us to identify proteins involved in endoplasmic reticulum (ER) QC of the receptor. Among ß2AR-associated proteins is Ring finger protein 5 (RNF5), an E3 ubiquitin ligase anchored to the outer membrane of the ER. Coimmunoprecipitation assays confirmed, in a cellular context, the interaction between RNF5 and the ß2AR as well as the prostaglandin D2 receptor (DP). Confocal microscopy revealed that DP colocalized with RNF5 at the ER. Coexpression of RNF5 with either receptor increased levels of their expression, whereas small interfering RNA-mediated knockdown of endogenous RNF5 promoted the opposite. RNF5 did not modulate the ubiquitination state of ß2AR or DP. Instead, RNF5 ubiquitinated JNK-associated membrane protein (JAMP), a protein that recruits the proteasome to the ER membrane and that is negatively regulated by RNF5-mediated ubiquitination. JAMP coimmunoprecipitated with both ß2AR and DP and decreased total receptor protein levels through proteasomal degradation. Expression of DP, a receptor largely retained in the ER, promoted proteasome recruitment by JAMP. Degradation of both receptors via JAMP was increased when RNF5 was depleted. Our data suggest that RNF5 regulates the turnover of specific G protein-coupled receptors by ubiquitinating JAMP and preventing proteasome recruitment.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico , Proteômica , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Both ß(1)- and ß(3)-adrenergic receptors (ß(1)ARs and ß(3)ARs) are present on nuclear membranes in adult ventricular myocytes. These nuclear-localized receptors are functional with respect to ligand binding and effector activation. In isolated cardiac nuclei, the non-selective ßAR agonist isoproterenol stimulated de novo RNA synthesis measured using assays of transcription initiation (Boivin et al., 2006 Cardiovasc Res. 71:69-78). In contrast, stimulation of endothelin receptors, another G protein-coupled receptor (GPCR) that localizes to the nuclear membrane, resulted in decreased RNA synthesis. To investigate the signalling pathway(s) involved in GPCR-mediated regulation of RNA synthesis, nuclei were isolated from intact adult rat hearts and treated with receptor agonists in the presence or absence of inhibitors of different mitogen-activated protein kinase (MAPK) and PI3K/PKB pathways. Components of p38, JNK, and ERK1/2 MAP kinase cascades as well as PKB were detected in nuclear preparations. Inhibition of PKB with triciribine, in the presence of isoproterenol, converted the activation of the ßAR from stimulatory to inhibitory with regards to RNA synthesis, while ERK1/2, JNK and p38 inhibition reduced both basal and isoproterenol-stimulated activity. Analysis by qPCR indicated an increase in the expression of 18S rRNA following isoproterenol treatment and a decrease in NFκB mRNA. Further qPCR experiments revealed that isoproterenol treatment also reduced the expression of several other genes involved in the activation of NFκB, while ERK1/2 and PKB inhibition substantially reversed this effect. Our results suggest that GPCRs on the nuclear membrane regulate nuclear functions such as gene expression and this process is modulated by activation/inhibition of downstream protein kinases within the nucleus.
Assuntos
Núcleo Celular/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Animais , Regulação da Expressão Gênica , Isoproterenol/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , RNA Ribossômico 18S/metabolismo , Ratos , Receptores Adrenérgicos beta/genética , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: Innovations in pediatric cardiovascular surgery have resulted in significant improvements in survival for children with congenital heart disease. In adults with such disease, however, surgical morbidity and mortality remain significant. We hypothesized that hypoxemia in early life causes lasting changes in gene expression in the developing heart and that such changes may persist into later life, affecting the physiology of the adult myocardium. METHODS: Microarray expression analyses were performed with left ventricular tissue from 10- and 90-day-old rats exposed to hypoxia (inspired oxygen fraction 0.12) for the first 10 days after birth then subsequently reared in ambient air and with tissue from age-matched rats reared entirely in ambient air. Changes in expression of selected genes were confirmed with real-time reverse transcriptase polymerase chain reaction. Left ventricular cardiomyocytes were isolated from adult animals in both groups, and cellular morphology and viability were compared. RESULTS: Microarray analyses revealed significant changes in 1945 and 422 genes in neonates and adults, respectively. Changes in genes associated with adaptive vascular remodeling and energy homeostasis, as well as regulation of apoptosis, were confirmed by real-time reverse transcriptase polymerase chain reaction. The viability of cardiomyocytes isolated from hypoxic animals was significantly lower than in those from control animals (36.7% +/- 13.3% vs 85.0% +/- 2.9%, P = .024). CONCLUSIONS: Neonatal hypoxia is associated with significant changes in left ventricular gene expression in both neonatal and adult rats. This may have physiologic implications for the adult myocardium.
Assuntos
Angiopoietinas/metabolismo , Asfixia Neonatal/genética , Modelos Animais de Doenças , Ventrículos do Coração/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Miocárdio/metabolismo , Receptor Notch1/metabolismo , Adaptação Fisiológica , Proteína 4 Semelhante a Angiopoietina , Animais , Animais Recém-Nascidos , Proteínas Reguladoras de Apoptose , Biomarcadores Tumorais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Perfilação da Expressão Gênica , Transportador de Glucose Tipo 4/metabolismo , Humanos , Recém-Nascido , Isoenzimas/metabolismo , L-Lactato Desidrogenase/metabolismo , Lactato Desidrogenase 5 , Masculino , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosfoproteínas/metabolismo , Fosfopiruvato Hidratase/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/metabolismo , Regulação para CimaRESUMO
Bioluminescence resonance energy transfer (BRET) and co-immunoprecipitation experiments revealed that heterotrimeric G proteins and their effectors were found in stable complexes that persisted during signal transduction. Adenylyl cyclase, Kir3.1 channel subunits and several G-protein subunits (Galpha(s), Galpha(i), Gbeta(1) and Ggamma(2)) were tagged with luciferase (RLuc) or GFP, or the complementary fragments of YFP (specifically Gbeta(1)-YFP(1-158) and Ggamma(2)-YFP(159-238), which heterodimerize to produce fluorescent YFP-Gbeta(1)gamma(2)). BRET was observed between adenylyl-cyclase-RLuc or Kir3.1-RLuc and GFP-Ggamma(2), GFP-Gbeta(1) or YFP-Gbeta(1)gamma(2). Galpha subunits were also stably associated with both effectors regardless of whether or not signal transduction was initiated by a receptor agonist. Although BRET between effectors and Gbetagamma was increased by receptor stimulation, our data indicate that these changes are likely to be conformational in nature. Furthermore, receptor-sensitive G-protein-effector complexes could be detected before being transported to the plasma membrane, providing the first direct evidence for an intracellular site of assembly.