RESUMEN
BACKGROUND: B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T-cell therapies have generated responses in patients with advanced myeloma, but relapses are common. G protein-coupled receptor, class C, group 5, member D (GPRC5D) has been identified as an immunotherapeutic target in multiple myeloma. Preclinical studies have shown the efficacy of GPRC5D-targeted CAR T cells, including activity in a BCMA antigen escape model. METHODS: In this phase 1 dose-escalation study, we administered a GPRC5D-targeted CAR T-cell therapy (MCARH109) at four dose levels to patients with heavily pretreated multiple myeloma, including patients with relapse after BCMA CAR T-cell therapy. RESULTS: A total of 17 patients were enrolled and received MCARH109 therapy. The maximum tolerated dose was identified at 150×106 CAR T cells. At the 450×106 CAR T-cell dose, 1 patient had grade 4 cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome (ICANS), and 2 patients had a grade 3 cerebellar disorder of unclear cause. No cerebellar disorder, ICANS of any grade, or cytokine release syndrome of grade 3 or higher occurred in the 12 patients who received doses of 25×106 to 150×106 cells. A response was reported in 71% of the patients in the entire cohort and in 58% of those who received doses of 25×106 to 150×106 cells. The patients who had a response included those who had received previous BCMA therapies; responses were observed in 7 of 10 such patients in the entire cohort and in 3 of 6 such patients who received 25×106 to 150×106 cells. CONCLUSIONS: The results of this study of a GPRC5D-targeted CAR T-cell therapy (MCARH109) confirm that GPRC5D is an active immunotherapeutic target in multiple myeloma. (Funded by Juno Therapeutics/Bristol Myers Squibb; ClinicalTrials.gov number, NCT04555551.).
Asunto(s)
Inmunoterapia Adoptiva , Mieloma Múltiple , Receptores Quiméricos de Antígenos , Receptores Acoplados a Proteínas G , Antígeno de Maduración de Linfocitos B/uso terapéutico , Síndrome de Liberación de Citoquinas/etiología , Humanos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Mieloma Múltiple/tratamiento farmacológico , Recurrencia Local de Neoplasia/etiología , Receptores Quiméricos de Antígenos/uso terapéutico , Receptores Acoplados a Proteínas G/uso terapéutico , Linfocitos TRESUMEN
RATIONALE: Despite overwhelming evidence of the importance of circadian rhythms in cardiovascular health and disease, little is known regarding the circadian regulation of intracellular signaling pathways controlling cardiac function and remodeling. OBJECTIVE: To assess circadian changes in processes dependent on the protein phosphatase calcineurin, relative to changes in phosphorylation of cardiac proteins, in normal, hypertrophic, and failing hearts. METHODS AND RESULTS: We found evidence of large circadian oscillations in calcineurin-dependent activities in the left ventricle of healthy C57BL/6 mice. Calcineurin-dependent transcript levels and nuclear occupancy of the NFAT (nuclear factor of activated T cells) regularly fluctuated as much as 20-fold over the course of a day, peaking in the morning when mice enter a period of rest. Phosphorylation of the protein phosphatase 1 inhibitor 1 (I-1), a direct calcineurin substrate, and phospholamban, an indirect target, oscillated directly out of phase with calcineurin-dependent signaling. Using a surgical model of cardiac pressure overload, we found that although calcineurin-dependent activities were markedly elevated, the circadian pattern of activation was maintained, whereas, oscillations in phospholamban and I-1 phosphorylation were lost. Changes in the expression of fetal gene markers of heart failure did not mirror the rhythm in calcineurin/NFAT activation, suggesting that these may not be direct transcriptional target genes. Cardiac function in mice subjected to pressure overload was significantly lower in the morning than in the evening when assessed by echocardiography. CONCLUSIONS: Normal, opposing circadian oscillations in calcineurin-dependent activities and phosphorylation of proteins that regulate contractility are disrupted in heart failure.
Asunto(s)
Calcineurina/fisiología , Ritmo Circadiano/fisiología , Insuficiencia Cardíaca/metabolismo , Hemodinámica/fisiología , Proteínas/metabolismo , Transducción de Señal/fisiología , Estrés Fisiológico/fisiología , Animales , Proteínas de Unión al Calcio/metabolismo , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/fisiopatología , Ventrículos Cardíacos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Musculares/metabolismo , Factores de Transcripción NFATC/metabolismo , Fosforilación/fisiología , Proteína Fosfatasa 1/metabolismoRESUMEN
With the US Food and Drug Administration (FDA) approval of four CD19- and one BCMA-targeted chimeric antigen receptor (CAR) therapy for B cell malignancies, CAR T cell therapy has finally reached the status of a medicinal product. The successful manufacturing of autologous CAR T cell products is a key requirement for this promising treatment modality. By analyzing the composition of 214 apheresis products from 210 subjects across eight disease indications, we found that high CD14+ cell content poses a challenge for manufacturing CAR T cells, especially in patients with non-Hodgkin's lymphoma and multiple myeloma caused by the non-specific phagocytosis of the magnetic beads used to activate CD3+ T cells. We demonstrated that monocyte depletion via rapid plastic surface adhesion significantly reduces the CD14+ monocyte content in the apheresis products and simultaneously boosts the CD3+ content. We established a 40% CD14+ threshold for the stratification of apheresis products across nine clinical trials and demonstrated the effectiveness of this procedure by comparing manufacturing runs in two phase 1 clinical trials. Our study suggests that CD14+ content should be monitored in apheresis products, and that the manufacturing of CAR T cells should incorporate a step that lessens the CD14+ cell content in apheresis products containing more than 40% to maximize the production success.
RESUMEN
Malignant pleural diseases, comprising metastatic lung and breast cancers and malignant pleural mesothelioma (MPM), are aggressive solid tumors with poor therapeutic response. We developed and conducted a first-in-human, phase I study of regionally delivered, autologous, mesothelin-targeted chimeric antigen receptor (CAR) T-cell therapy. Intrapleural administration of 0.3M to 60M CAR T cells/kg in 27 patients (25 with MPM) was safe and well tolerated. CAR T cells were detected in peripheral blood for >100 days in 39% of patients. Following our demonstration that PD-1 blockade enhances CAR T-cell function in mice, 18 patients with MPM also received pembrolizumab safely. Among those patients, median overall survival from CAR T-cell infusion was 23.9 months (1-year overall survival, 83%). Stable disease was sustained for ≥6 months in 8 patients; 2 exhibited complete metabolic response on PET scan. Combination immunotherapy with CAR T cells and PD-1 blockade agents should be further evaluated in patients with solid tumors. SIGNIFICANCE: Regional delivery of mesothelin-targeted CAR T-cell therapy followed by pembrolizumab administration is feasible, safe, and demonstrates evidence of antitumor efficacy in patients with malignant pleural diseases. Our data support the investigation of combination immunotherapy with CAR T cells and PD-1 blockade agents in solid tumors.See related commentary by Aldea et al., p. 2674.This article is highlighted in the In This Issue feature, p. 2659.
Asunto(s)
Mesotelioma , Enfermedades Pleurales , Anticuerpos Monoclonales Humanizados , Humanos , Inmunoterapia Adoptiva , Mesotelina , Mesotelioma/tratamiento farmacológicoRESUMEN
Although it is a widely studied psychiatric syndrome, major depressive disorder remains a poorly understood illness, especially with regard to the disconnect between treatment initiation and the delayed onset of clinical improvement. We have recently validated chronic social defeat stress in mice as a model in which a depression-like phenotype is reversed by chronic, but not acute, antidepressant administration. Here, we use chromatin immunoprecipitation (ChIP)-chip assays--ChIP followed by genome wide promoter array analyses--to study the effects of chronic defeat stress on chromatin regulation in the mouse nucleus accumbens (NAc), a key brain reward region implicated in depression. Our results demonstrate that chronic defeat stress causes widespread and long-lasting changes in gene regulation, including alterations in repressive histone methylation and in phospho-CREB (cAMP response element-binding protein) binding, in the NAc. We then show similarities and differences in this regulation to that observed in another mouse model of depression, prolonged adult social isolation. In the social defeat model, we observed further that many of the stress-induced changes in gene expression are reversed by chronic imipramine treatment, and that resilient mice-those resistant to the deleterious effects of defeat stress-show patterns of chromatin regulation in the NAc that overlap dramatically with those seen with imipramine treatment. These findings provide new insight into the molecular basis of depression-like symptoms and the mechanisms by which antidepressants exert their delayed clinical efficacy. They also raise the novel idea that certain individuals resistant to stress may naturally mount antidepressant-like adaptations in response to chronic stress.
Asunto(s)
Antidepresivos Tricíclicos/uso terapéutico , Cromatina/efectos de los fármacos , Depresión/tratamiento farmacológico , Depresión/patología , Imipramina/uso terapéutico , Núcleo Accumbens/ultraestructura , Animales , Conducta Animal , Proteína de Unión a CREB/metabolismo , Inmunoprecipitación de Cromatina/métodos , Modelos Animales de Enfermedad , Dominación-Subordinación , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Estudio de Asociación del Genoma Completo/métodos , Histonas/metabolismo , Masculino , Metilación/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Núcleo Accumbens/efectos de los fármacos , Aislamiento SocialRESUMEN
Sug1 and Sug2 are two of six ATPases in the 19S regulatory particle of the 26S proteasome. We have shown previously that these proteins play a non-proteolytic role in the transcription of the GAL genes in yeast. In this study, we probe the requirement for these factors in stress-induced transcription in yeast. It is known that proteasomal proteolysis is not required for these events. Indeed, proteasome inhibitors strongly stimulate expression of these stress response genes. However, shifting strains carrying temperature-sensitive alleles of SUG1 and SUG2 to the restrictive temperature strongly inhibited the expression of HSP26, HSP104 and GAD1 in response to heat shock or treatment with menadione bisulfate. Furthermore, chromatin immunoprecipitation analysis revealed the recruitment of Sug1, Sug2 and Cim5 (another of the ATPases), but not 20S proteasome core proteins, to the promoters of these genes. These data show that the non-proteolytic requirement for the proteasomal ATPases extends beyond the GAL genes in yeast and includes at least the heat and oxidative stress-responsive genes.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Regulación Fúngica de la Expresión Génica , Proteínas Represoras/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , ADN Polimerasa III/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/genética , Respuesta al Choque Térmico , Estrés Oxidativo , Regiones Promotoras Genéticas , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
A central issue in the regulation of gene expression is the physical association of transcription factors with relevant promoter sequences. Recently, technological advances have allowed researchers to analyze these processes on a genomic scale. In particular, the combination of the chromatin immunoprecipitation (ChIP) technique with microarray analysis (the 'ChIP to chip' experiment) is providing a wealth of new and surprising data on transcription factor-chromatin interactions. These advances are reviewed here. We also discuss future challenges in the area.
Asunto(s)
Inmunoprecipitación de Cromatina , ADN/metabolismo , Genómica , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Biología Computacional/métodos , ADN/química , Regulación de la Expresión Génica , Humanos , Factores de Transcripción/químicaRESUMEN
Although HSF1 plays an important role in the cellular response to proteotoxic stressors, little is known about the structure and function of the human HSF1 signaling network under both stressed and unstressed conditions. In this study, we used a combination of chromatin immunoprecipitation microarray analysis and time course gene expression microarray analysis with and without siRNA-mediated inhibition of HSF1 to comprehensively identify genes regulated directly and indirectly by HSF1. The correlation between promoter binding and gene expression was not significant for all genes bound by HSF1, suggesting that HSF1 binding per se is not sufficient for expression. However, the correlation with promoter binding was significant for genes identified as HSF1-regulated following siRNA knockdown. Among promoters bound by HSF1 following heat shock, a gene ontology analysis showed significant enrichment only in categories related to protein folding. In contrast, analysis of the extended HSF1 signaling network following siRNA knockdown showed enrichment in a variety of categories related to protein folding, anti-apoptosis, RNA splicing, ubiquitinylation and others, highlighting a complex transcriptional program regulated directly and indirectly by HSF1.
Asunto(s)
Adaptación Fisiológica , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Supervivencia Celular , Inmunoprecipitación de Cromatina , Genoma Humano , Células HeLa , Factores de Transcripción del Choque Térmico , Respuesta al Choque Térmico , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Factores de Tiempo , TransfecciónRESUMEN
The aging process causes an increase in percent body fat, but the mechanism remains unclear. In the present study we examined the impact of aging on brown adipose tissue (BAT) thermogenic activity as potential cause for the increase in adiposity. We show that aging is associated with interscapular BAT morphologic abnormalities and thermogenic dysfunction. In vitro experiments revealed that brown adipocyte differentiation is defective in aged mice. Interscapular brown tissue in aged mice is progressively populated by adipocytes bearing white morphologic characteristics. Aged mice fail to mobilize intracellular fuel reserves from brown adipocytes and exhibit deficiency in homeothermy. Our results suggest a role for orexin (OX) signaling in the regulation of thermogenesis during aging. Brown fat dysfunction and age-related assimilation of fat mass were accelerated in mice in which OX-producing neurons were ablated. Conversely, OX injections in old mice increased multilocular morphology, increased core body temperature, improved cold tolerance, and reduced adiposity. These results argue that BAT can be targeted for interventions to reverse age-associated increase in fat mass.
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Adipocitos/efectos de los fármacos , Tejido Adiposo Pardo/efectos de los fármacos , Envejecimiento/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/farmacología , Neuropéptidos/farmacología , Termogénesis/efectos de los fármacos , Adipocitos/metabolismo , Tejido Adiposo Pardo/metabolismo , Adiposidad/efectos de los fármacos , Adiposidad/fisiología , Envejecimiento/metabolismo , Animales , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/fisiología , Masculino , Ratones , Orexinas , Termogénesis/fisiologíaRESUMEN
Orexins are a pair of hypothalamic neuropeptides that were discovered in the late 1990s and named initially for their ability to promote feeding. Subsequent studies have revealed the importance of orexins to a variety of physiological functions, including brown fat thermogenesis, sleep/wake cycles, physical activity, and cognition. We aim to elucidate the various roles of orexins and discuss how these multiple functions are interlinked. We explain that although the unique dual roles of orexins in increasing feeding while concomitantly elevating energy expenditure appear counterproductive, they are necessary for physiological scenarios during which simultaneous stimulation of energy expenditure and feeding occur, namely diet-induced thermogenesis and arousal from hibernation. The position of orexins at the interface between sleep/wake cycles, energy homeostasis, and environmental factors has important implications in the treatment of obesity.
Asunto(s)
Hipotálamo/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neuropéptidos/metabolismo , Tejido Adiposo Pardo/fisiología , Nivel de Alerta/fisiología , Ingestión de Alimentos , Humanos , Orexinas , Sueño/fisiologíaRESUMEN
Nutrition plays a dominant role in human adaptation. Biological traits conferring these adaptations are of considerable significance. Within an obesogenic environment, there is considerable variation among individuals in their susceptibility to weight gain. Some individuals rapidly gain weight, whereas others remain lean without any conscious effort, suggesting that obesity pathogenesis may not be centered on just the primal feeding behavior. The ability of certain individuals to subconsciously resist obesity reveals adaptive calorie-burning mechanisms that may promote fitness. Here, we review a fat-burning mechanism that is turned on by the brain hormone orexin during high-caloric food consumption. Remarkably, the same hormone also induces feeding, and its levels correlate with lean body mass in both rodents and humans. Intriguingly, loss of orexin prevents thermogenic energy expenditure while inducing obesity in the face of hypophagia. Thus, orexin is a unique neuropeptide that promotes both feeding and energy expenditure, conferring resistance to weight gain. Mechanisms that safely augment orexin signaling may have potential in antiobesity therapeutics.
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Tejido Adiposo Pardo/fisiología , Conducta Alimentaria/fisiología , Obesidad/etiología , Termogénesis , Tejido Adiposo Pardo/metabolismo , Adiposidad/genética , Animales , Metabolismo Energético/genética , Metabolismo Energético/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Narcolepsia/etiología , Narcolepsia/genética , Narcolepsia/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismo , Neuropéptidos/fisiología , Obesidad/genética , Obesidad/metabolismo , Orexinas , Sueño/genética , Sueño/fisiología , Termogénesis/efectos de los fármacos , Vigilia/genética , Vigilia/fisiologíaAsunto(s)
Factores de Transcripción/síntesis química , Secuencia de Bases , Técnicas Químicas Combinatorias , ADN/química , ADN/metabolismo , Expresión Génica/efectos de los fármacos , Genes Reporteros/genética , Células HeLa , Humanos , Imitación Molecular , Datos de Secuencia Molecular , Nylons/químicaRESUMEN
Orexin A (OX) is a small excitatory neuropeptide hormone that stimulates feeding, wakefulness and energy expenditure via a pair of G-coupled protein receptors, namely orexin receptor-1 (OXR1) and orexin receptor-2 (OXR2). OX-deficient mice are sensitive to obesity despite being hypophagic. The obesogenic effect of OX-deletion is due to brown adipose tissue (BAT) dysfunction, a defect that originates during fetal growth. Brown preadipocytes in OX-null mice display undifferentiated histological appearance and fail to support both diet- and cold-induced thermogenesis. We show that the OXR1-null mice phenocopies the differentiation defect observed in the ligand-null mice indicating that OXR1 relays OX's differentiation and thermogenic function. Consistent with this, OX fails to induce differentiation in cultured OXR1-null preadipocytes. Thus, OX signaling via OXR1 constitutes an important thermoregulatory mechanism that defends against cold and obesity.
RESUMEN
Orexin (OX) neuropeptides stimulate feeding and arousal. Deficiency of orexin is implicated in narcolepsy, a disease associated with obesity, paradoxically in the face of reduced food intake. Here, we show that obesity in orexin-null mice is associated with impaired brown adipose tissue (BAT) thermogenesis. Failure of thermogenesis in OX-null mice is due to inability of brown preadipocytes to differentiate. The differentiation defect in OX-null neonates is circumvented by OX injections to OX-null dams. In vitro, OX, triggers the full differentiation program in mesenchymal progenitor stem cells, embryonic fibroblasts and brown preadipocytes via p38 mitogen activated protein (MAP) kinase and bone morphogenetic protein receptor-1a (BMPR1A)-dependent Smad1/5 signaling. Our study suggests that obesity associated with OX depletion is linked to brown-fat hypoactivity, which leads to dampening of energy expenditure. Thus, orexin plays an integral role in adaptive thermogenesis and body weight regulation via effects on BAT differentiation and function.
Asunto(s)
Tejido Adiposo Pardo/citología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neuropéptidos/metabolismo , Tejido Adiposo Pardo/metabolismo , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/antagonistas & inhibidores , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Diferenciación Celular , Línea Celular , Metabolismo Energético/fisiología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuropéptidos/antagonistas & inhibidores , Neuropéptidos/genética , Obesidad/etiología , Orexinas , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Termogénesis/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Cardiac hypertrophy develops in response to a variety of cardiovascular stresses and results in activation of numerous signaling cascades and proteins. In the present study, we demonstrate that cytoglobin is a stress-responsive hemoprotein in the hypoxia-induced hypertrophic myocardium and it is transcriptionally regulated by calcineurin-dependent transcription factors. The cytoglobin transcript level is abundantly expressed in the adult heart and in response to hypoxia cytoglobin expression is markedly up-regulated within the hypoxia-induced hypertrophic heart. To define the molecular mechanism resulting in the induction of cytoglobin, we undertook a transcriptional analysis of the 5' upstream regulatory region of the cytoglobin gene. Evolutionarily conserved binding elements for transcription factors HIF-1, AP-1, and NFAT are located within the upstream region of the cytoglobin gene. Transcriptional assays demonstrated that calcineurin activity modulates cytoglobin transcription. Increased calcineurin activity enhances the ability of NFAT and AP-1 to bind to the putative cytoglobin promoter, especially under hypoxic conditions. In addition, inhibition of calcineurin, NFAT, and/or AP-1 activities decreases endogenous cytoglobin transcript and protein levels. Thus, the regulation of cytoglobin transcription by calcineurin-dependent transcription factors suggests that cytoglobin may have a functional role in calcium-dependent events accompanying cardiac remodeling.
Asunto(s)
Calcineurina/metabolismo , Globinas/metabolismo , Hipoxia , Miocitos Cardíacos/fisiología , Activación Transcripcional , Animales , Secuencia de Bases , Calcineurina/genética , Línea Celular , Citoglobina , Globinas/genética , Humanos , Hipertrofia Ventricular Izquierda/metabolismo , Hipoxia/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutagénesis Sitio-Dirigida , Miocitos Cardíacos/citología , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Elementos de Respuesta/genética , Alineación de SecuenciaRESUMEN
Changes in gene expression contribute to the long-lasting regulation of the brain's reward circuitry seen in drug addiction; however, the specific genes regulated and the transcriptional mechanisms underlying such regulation remain poorly understood. Here, we used chromatin immunoprecipitation coupled with promoter microarray analysis to characterize genome-wide chromatin changes in the mouse nucleus accumbens, a crucial brain reward region, after repeated cocaine administration. Our findings reveal several interesting principles of gene regulation by cocaine and of the role of DeltaFosB and CREB, two prominent cocaine-induced transcription factors, in this brain region. The findings also provide comprehensive insight into the molecular pathways regulated by cocaine-including a new role for sirtuins (Sirt1 and Sirt2)-which are induced in the nucleus accumbens by cocaine and, in turn, dramatically enhance the behavioral effects of the drug.
Asunto(s)
Cromatina/efectos de los fármacos , Cocaína/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Núcleo Accumbens/efectos de los fármacos , Sirtuinas/metabolismo , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Inhibidores de Captación de Dopamina/farmacología , Retroalimentación Fisiológica/efectos de los fármacos , Estudio de Asociación del Genoma Completo , Masculino , Ratones , Ratones Endogámicos C57BL , Núcleo Accumbens/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/fisiología , Proteínas Proto-Oncogénicas c-fos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Refuerzo en Psicología , Transducción de Señal/efectos de los fármacos , Sirtuinas/efectos de los fármacosRESUMEN
Recent analysis of a Gal4 mutant (Gap71) carrying three point mutations (S22D, K23Q and K25F) in its DNA-binding domain (DBD), has demonstrated that it cannot occupy GAL promoters efficiently in cells and that it is not mono-ubiquitylated, suggesting a functional link between this modification and stable DNA binding in cells. The mechanistic underpinning of this phenotype is that this protein is hypersensitive to a newly discovered activity of the proteasomal ATPases--their ability to actively dissociate transcription factor-DNA complexes after direct interaction with the activation domain. In this paper, we examine the roles of each of the three point mutations contained in Gap71 individually. These experiments have revealed that serine 22 is a site of phosphorylation in the Gal4 DBD and that lysine 23 is essential for S22 phosphorylation, possibly acting as part of the kinase recognition site. Mutation of either residue blocks Gal4 DBD phosphorylation, its subsequent ubiquitylation and compromises the ability of the activator to bind promoter DNA in vivo. These data represent the first report of an essential phosphorylation event that is critical for the activity of this paradigmatic transcription factor.
Asunto(s)
ADN/metabolismo , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Proteínas de Unión al ADN , Células HeLa , Humanos , Lisina/genética , Lisina/metabolismo , Modelos Moleculares , Fosforilación , Mutación Puntual , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Serina/genética , Serina/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , UbiquitinaciónRESUMEN
Repeated exposure to cocaine causes sensitized behavioral responses and increased dendritic spines on medium spiny neurons of the nucleus accumbens (NAc). We find that cocaine regulates myocyte enhancer factor 2 (MEF2) transcription factors to control these two processes in vivo. Cocaine suppresses striatal MEF2 activity in part through a mechanism involving cAMP, the regulator of calmodulin signaling (RCS), and calcineurin. We show that reducing MEF2 activity in the NAc in vivo is required for the cocaine-induced increases in dendritic spine density. Surprisingly, we find that increasing MEF2 activity in the NAc, which blocks the cocaine-induced increase in dendritic spine density, enhances sensitized behavioral responses to cocaine. Together, our findings implicate MEF2 as a key regulator of structural synapse plasticity and sensitized responses to cocaine and suggest that reducing MEF2 activity (and increasing spine density) in NAc may be a compensatory mechanism to limit long-lasting maladaptive behavioral responses to cocaine.
Asunto(s)
Cocaína/farmacología , Espinas Dendríticas/efectos de los fármacos , Inhibidores de Captación de Dopamina/farmacología , Factores Reguladores Miogénicos/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Núcleo Accumbens/efectos de los fármacos , Adaptación Fisiológica/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Quinasas Ciclina-Dependientes/efectos de los fármacos , Regulación hacia Abajo , Esquema de Medicación , Perfilación de la Expresión Génica , Factores de Transcripción MEF2 , Masculino , Ratones , Ratones Endogámicos C57BL , Neostriado/citología , Neostriado/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Ratas Long-Evans , Ratas Sprague-Dawley , Sinapsis/efectos de los fármacosRESUMEN
Orexin A and Orexin B (also known as hypocretins) are neuropeptides that bind two related G-coupled protein receptors (OXR1 and OXR2) and thus induce wakefulness, food consumption, and locomotion. Conversely, deletion of the orexin gene in mice produces a condition similar to canine and human narcolepsy. Despite the central importance of the orexin system in regulating wakefulness and feeding behavior, little is known about the downstream signaling mechanisms that achieve these effects. In this study, genomics techniques are used to probe this question and reveal that orexin activates the hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor whose pathogenic role in stimulating angiogenesis in hypoxic tumors has been the focus of intense investigation. Orexin-stimulated HIF-1 activity is due to both increased HIF-1alpha gene transcription and a down-regulation of von Hippel-Lindau (VHL), the E3 ubiquitin ligase that mediates the turnover of HIF-1 via the ubiquitin-proteasome pathway. Orexin-mediated activation of HIF-1 results in increased glucose uptake and higher glycolytic activity, as expected from studies of hypoxic cells. However, orexin receptor-expressing cells somehow override the HIF-1-mediated preference for funneling pyruvate into anaerobic glycolysis and instead favor ATP production through the tricarboxylic acid cycle and oxidative phosphorylation. These findings implicate HIF-1 as an important transcription factor in the hormone-mediated regulation of hunger and wakefulness.
Asunto(s)
Factor 1 Inducible por Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/farmacología , Neuropéptidos/farmacología , Neurotransmisores/farmacología , Adenosina Trifosfato/análisis , Animales , Técnicas de Cultivo de Célula , Línea Celular , Inmunoprecipitación de Cromatina , Cobalto/farmacología , Medio de Cultivo Libre de Suero , Genes Reporteros , Glucosa/metabolismo , Humanos , Hipotálamo/citología , Riñón/citología , Ácido Láctico/análisis , Luciferasas de Luciérnaga/metabolismo , Ratones , Ratones Noqueados , Proteínas Mitocondriales , Receptores de Orexina , Orexinas , Técnicas de Cultivo de Órganos , Plásmidos , Proteínas/metabolismo , Piruvato Deshidrogenasa (Lipoamida)-Fosfatasa/análisis , Receptores de Neuropéptido/genética , Receptores de Neuropéptido/metabolismo , TransfecciónRESUMEN
Recent studies have shown that the intersection between transcription and proteins involved in the ubiquitin-proteasome pathway encompasses both proteolytic and nonproteolytic functions. Examples of the latter type include evidence that monoubiquitylation of some transcriptional activators stimulates their activity. In addition, the proteasomal ATPases are recruited to many active promoters through binding to activators and play an important, nonproteolytic role in promoter escape and elongation. In this study, we report the discovery of a new nonproteolytic activity of the proteasome (specifically the proteasomal ATPases): the active destabilization of activator-promoter complexes. This reaction depends on the presence of an activation domain and ATP. Destabilization is inhibited in vitro and in vivo if the protein is monoubiquitylated or if ubiquitin is genetically fused to the activator. The fact that monoubiquitylated activator is resistant to the "stripping" activity of the proteasomal ATPases may explain, in part, why some activators require this modification in order to function efficiently.