RESUMEN
Despite huge efforts made in academic and pharmaceutical worldwide research, current anticancer therapies achieve effective treatment in a limited number of neoplasia cases only. Oncology terms such as big killers - to identify tumours with yet a high mortality rate - or undruggable cancer targets, and chemoresistance, represent the current therapeutic debacle of cancer treatments. In addition, metastases, tumour microenvironments, tumour heterogeneity, metabolic adaptations, and immunotherapy resistance are essential features controlling tumour response to therapies, but still, lack effective therapeutics or modulators. In this scenario, where the pharmaceutical productivity and drug efficacy in oncology seem to have reached a plateau, the so-called drug repurposing - i.e. the use of old drugs, already in clinical use, for a different therapeutic indication - is an appealing strategy to improve cancer therapy. Opportunities for drug repurposing are often based on occasional observations or on time-consuming pre-clinical drug screenings that are often not hypothesis-driven. In contrast, in-silico drug repurposing is an emerging, hypothesis-driven approach that takes advantage of the use of big-data. Indeed, the extensive use of -omics technologies, improved data storage, data meaning, machine learning algorithms, and computational modeling all offer unprecedented knowledge of the biological mechanisms of cancers and drugs' modes of action, providing extensive availability for both disease-related data and drugs-related data. This offers the opportunity to generate, with time and cost-effective approaches, computational drug networks to predict, in-silico, the efficacy of approved drugs against relevant cancer targets, as well as to select better responder patients or disease' biomarkers. Here, we will review selected disease-related data together with computational tools to be exploited for the in-silico repurposing of drugs against validated targets in cancer therapies, focusing on the oncogenic signaling pathways activation in cancer. We will discuss how in-silico drug repurposing has the promise to shortly improve our arsenal of anticancer drugs and, likely, overcome certain limitations of modern cancer therapies against old and new therapeutic targets in oncology.
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Antineoplásicos/uso terapéutico , Diseño de Fármacos/métodos , Descubrimiento de Drogas , Reposicionamiento de Medicamentos/métodos , Neoplasias/tratamiento farmacológico , Animales , HumanosRESUMEN
Oncogenic v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-RAS) plays a key role in the development and maintenance of pancreatic ductal adenocarcinoma (PDAC). The targeting of K-RAS would be beneficial to treat tumors whose growth depends on active K-RAS. The analysis of K-RAS genomic mutations is a clinical routine; however, an emerging question is whether the mutational status is able to identify tumors effectively dependent on K-RAS for tailoring targeted therapies. With the emergence of novel K-RAS inhibitors in clinical settings, this question is relevant. Several studies support the notion that the K-RAS mutation is not a sufficient biomarker deciphering the effective dependency of the tumor. Transcriptomic and metabolomic profiles of tumors, while revealing K-RAS signaling complexity and K-RAS-driven molecular pathways crucial for PDAC growth, are opening the opportunity to specifically identify K-RAS-dependent- or K-RAS-independent tumor subtypes by using novel molecular biomarkers. This would help tumor selection aimed at tailoring therapies against K-RAS. In this review, we will present studies about how the K-RAS mutation can also be interpreted in a state of K-RAS dependency, for which it is possible to identify specific K-RAS-driven molecular biomarkers in certain PDAC subtypes, beyond the genomic K-RAS mutational status.
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Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/patología , Humanos , Mutación , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patología , TranscriptomaRESUMEN
Cancer stem-like cells (CSC) induce aggressive tumor phenotypes such as metastasis formation, which is associated with poor prognosis in triple-negative breast cancer (TNBC). Repurposing of FDA-approved drugs that can eradicate the CSC subcompartment in primary tumors may prevent metastatic disease, thus representing an effective strategy to improve the prognosis of TNBC. Here, we investigated spheroid-forming cells in a metastatic TNBC model. This strategy enabled us to specifically study a population of long-lived tumor cells enriched in CSCs, which show stem-like characteristics and induce metastases. To repurpose FDA-approved drugs potentially toxic for CSCs, we focused on pyrvinium pamoate (PP), an anthelmintic drug with documented anticancer activity in preclinical models. PP induced cytotoxic effects in CSCs and prevented metastasis formation. Mechanistically, the cell killing effects of PP were a result of inhibition of lipid anabolism and, more specifically, the impairment of anabolic flux from glucose to cholesterol and fatty acids. CSCs were strongly dependent upon activation of lipid biosynthetic pathways; activation of these pathways exhibited an unfavorable prognostic value in a cohort of breast cancer patients, where it predicted high probability of metastatic dissemination and tumor relapse. Overall, this work describes a new approach to target aggressive CSCs that may substantially improve clinical outcomes for patients with TNBC, who currently lack effective targeted therapeutic options. SIGNIFICANCE: These findings provide preclinical evidence that a drug repurposing approach to prevent metastatic disease in TNBC exploits lipid anabolism as a metabolic vulnerability against CSCs in primary tumors.
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Antineoplásicos/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Compuestos de Pirvinio/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Colesterol/metabolismo , Reposicionamiento de Medicamentos , Femenino , Glucosa/metabolismo , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Ratones Endogámicos NOD , Células Madre Neoplásicas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoAsunto(s)
Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Reposicionamiento de Medicamentos , Neumonía Viral/tratamiento farmacológico , COVID-19 , Infecciones por Coronavirus/virología , Humanos , Pandemias , Neumonía Viral/virología , SARS-CoV-2 , Tratamiento Farmacológico de COVID-19RESUMEN
Mutated KRAS protein is a pivotal tumor driver in pancreatic cancer. However, despite comprehensive efforts, effective therapeutics that can target oncogenic KRAS are still under investigation or awaiting clinical approval. Using a specific KRAS-dependent gene signature, we implemented a computer-assisted inspection of a drug-gene network to in silico repurpose drugs that work like inhibitors of oncogenic KRAS. We identified and validated decitabine, an FDA-approved drug, as a potent inhibitor of growth in pancreatic cancer cells and patient-derived xenograft models that showed KRAS dependency. Mechanistically, decitabine efficacy was linked to KRAS-driven dependency on nucleotide metabolism and its ability to specifically impair pyrimidine biosynthesis in KRAS-dependent tumors cells. These findings also showed that gene signatures related to KRAS dependency might be prospectively used to inform on decitabine sensitivity in a selected subset of patients with KRAS-mutated pancreatic cancer. Overall, the repurposing of decitabine emerged as an intriguing option for treating pancreatic tumors that are addicted to mutant KRAS, thus offering opportunities for improving the arsenal of therapeutics for this extremely deadly disease. SIGNIFICANCE: Decitabine is a promising drug for cancer cells dependent on RAS signaling.
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Adenocarcinoma/tratamiento farmacológico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Decitabina/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Adenocarcinoma/metabolismo , Animales , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Reposicionamiento de Medicamentos/métodos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mutación/efectos de los fármacos , Neoplasias Pancreáticas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Glioblastoma Multiforme is the deadliest type of brain tumor and is characterized by very poor prognosis with a limited overall survival. Current optimal therapeutic approach has essentially remained unchanged for more than a decade, consisting in maximal surgical resection followed by radiotherapy plus temozolomide. MAIN BODY: Such a dismal patient outcome represents a compelling need for innovative and effective therapeutic approaches. Given the development of new drugs is a process presently characterized by an immense increase in costs and development time, drug repositioning, finding new uses for existing approved drugs or drug repurposing, re-use of old drugs when novel molecular findings make them attractive again, are gaining significance in clinical pharmacology, since it allows faster and less expensive delivery of potentially useful drugs from the bench to the bedside. This is quite evident in glioblastoma, where a number of old drugs is now considered for clinical use, often in association with the first-line therapeutic intervention. Interestingly, most of these medications are, or have been, widely employed for decades in non-neoplastic pathologies without relevant side effects. Now, the refinement of their molecular mechanism(s) of action through up-to-date technologies is paving the way for their use in the therapeutic approach of glioblastoma as well as other cancer types. SHORT CONCLUSION: The spiraling costs of new antineoplastic drugs and the long time required for them to reach the market demands a profoundly different approach to keep lifesaving therapies affordable for cancer patients. In this context, repurposing can represent a relatively inexpensive, safe and fast approach to glioblastoma treatment. To this end, pros and cons must be accurately considered.
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Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Antineoplásicos/química , Antineoplásicos/farmacología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Ensayos Clínicos como Asunto , Reposicionamiento de Medicamentos , Metabolismo Energético/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Relación Estructura-ActividadRESUMEN
The discovery of inhibitors for oncogenic signalling pathways remains a key focus in modern oncology, based on personalized and targeted therapeutics. Computational drug repurposing via the analysis of FDA-approved drug network is becoming a very effective approach to identify therapeutic opportunities in cancer and other human diseases. Given that gene expression signatures can be associated with specific oncogenic mutations, we tested whether a "reverse" oncogene-specific signature might assist in the computational repositioning of inhibitors of oncogenic pathways. As a proof of principle, we focused on oncogenic PI3K-dependent signalling, a molecular pathway frequently driving cancer progression as well as raising resistance to anticancer-targeted therapies. We show that implementation of "reverse" oncogenic PI3K-dependent transcriptional signatures combined with interrogation of drug networks identified inhibitors of PI3K-dependent signalling among FDA-approved compounds. This led to repositioning of Niclosamide (Niclo) and Pyrvinium Pamoate (PP), two anthelmintic drugs, as inhibitors of oncogenic PI3K-dependent signalling. Niclo inhibited phosphorylation of P70S6K, while PP inhibited phosphorylation of AKT and P70S6K, which are downstream targets of PI3K. Anthelmintics inhibited oncogenic PI3K-dependent gene expression and showed a cytostatic effect in vitro and in mouse mammary gland. Lastly, PP inhibited the growth of breast cancer cells harbouring PI3K mutations. Our data indicate that drug repositioning by network analysis of oncogene-specific transcriptional signatures is an efficient strategy for identifying oncogenic pathway inhibitors among FDA-approved compounds. We propose that PP and Niclo should be further investigated as potential therapeutics for the treatment of tumors or diseases carrying the constitutive activation of the PI3K/P70S6K signalling axis.
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Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Biología Computacional , Reposicionamiento de Medicamentos , Glándulas Mamarias Animales/efectos de los fármacos , Niclosamida/uso terapéutico , Compuestos de Pirvinio/uso terapéutico , Animales , Neoplasias de la Mama/patología , Carcinogénesis , Línea Celular Tumoral , Aprobación de Drogas , Femenino , Humanos , Glándulas Mamarias Animales/patología , Ratones , Niclosamida/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Compuestos de Pirvinio/farmacología , Proteínas Quinasas S6 Ribosómicas 70-kDa/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
Ultradian oscillators are cyclically expressed genes with a period of less than 24h, found in the major signalling pathways. The Notch effector hairy and enhancer of split Hes genes are ultradian oscillators. The physiological signals that synchronise and entrain Hes oscillators remain poorly understood. We investigated whether cellular metabolism modulates Hes1 cyclic expression. We demonstrated that, in mouse myoblasts (C2C12), Hes1 oscillation depends on reactive oxygen species (ROS), which are generated by the mitochondria electron transport chain and by NADPH oxidases NOXs. In vitro, the regulation of Hes1 by ROS occurs via the calcium-mediated signalling. The modulation of Hes1 by ROS was relevant in vivo, since perturbing ROS homeostasis was sufficient to alter Medaka (Oryzias latipes) somitogenesis, a process that is dependent on Hes1 ultradian oscillation during embryo development. Moreover, in a Medaka model for human microphthalmia with linear skin lesions syndrome, in which mitochondrial ROS homeostasis was impaired, we documented important somitogenesis defects and the deregulation of Hes homologues genes involved in somitogenesis. Notably, both molecular and developmental defects were rescued by antioxidant treatments. Our studies provide the first evidence of a coupling between cellular redox metabolism and an ultradian biological oscillator with important pathophysiological implication for somitogenesis.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Relojes Biológicos/fisiología , Modelos Animales de Enfermedad , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/fisiología , Oryzias/genética , Especies Reactivas de Oxígeno/metabolismo , Somitos/patología , Animales , Antioxidantes/farmacología , Calcio/metabolismo , Células Cultivadas , Complejo III de Transporte de Electrones/metabolismo , Embrión no Mamífero/metabolismo , Humanos , Hibridación in Situ , Ratones , Microftalmía/metabolismo , Microftalmía/patología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mioblastos/citología , Mioblastos/metabolismo , NADPH Oxidasas/metabolismo , Oryzias/embriología , Transducción de Señal , Anomalías Cutáneas/metabolismo , Anomalías Cutáneas/patología , Somitos/embriología , Somitos/metabolismo , Síndrome , Factor de Transcripción HES-1RESUMEN
MOTIVATION: Identification of differential expressed genes has led to countless new discoveries. However, differentially expressed genes are only a proxy for finding dysregulated pathways. The problem is to identify how the network of regulatory and physical interactions rewires in different conditions or in disease. RESULTS: We developed a procedure named DINA (DIfferential Network Analysis), which is able to identify set of genes, whose co-regulation is condition-specific, starting from a collection of condition-specific gene expression profiles. DINA is also able to predict which transcription factors (TFs) may be responsible for the pathway condition-specific co-regulation. We derived 30 tissue-specific gene networks in human and identified several metabolic pathways as the most differentially regulated across the tissues. We correctly identified TFs such as Nuclear Receptors as their main regulators and demonstrated that a gene with unknown function (YEATS2) acts as a negative regulator of hepatocyte metabolism. Finally, we showed that DINA can be used to make hypotheses on dysregulated pathways during disease progression. By analyzing gene expression profiles across primary and transformed hepatocytes, DINA identified hepatocarcinoma-specific metabolic and transcriptional pathway dysregulation. AVAILABILITY: We implemented an on-line web-tool http://dina.tigem.it enabling the user to apply DINA to identify tissue-specific pathways or gene signatures. CONTACT: dibernardo@tigem.it SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Redes Reguladoras de Genes , Redes y Vías Metabólicas/genética , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Células Cultivadas , Biología Computacional/métodos , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Hepatocitos/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Ratones , Especificidad de Órganos , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Glucose is an essential fuel for cell survival and its availability limits aberrant cellular proliferation. We have hypothesized that specific cancer mutations regulate metabolic response(s) to glucose deprivation (GD). By means of somatic knock-in cellular models, we have analyzed the response to glucose deprivation in cells carrying the frequent (delE746-A750)EGFR, (G13D)KRAS or (E545K)PIK3CA cancer alleles. We demonstrate that, in mammary epithelial cells, glucose has an essential antioxidant function and that these cells are very sensitive to GD. Conversely, isogenic cells carrying the (delE746-A750)EGFR or the (E545K)PIK3CA, but not the (G13D)KRAS allele, display high tolerance to GD by stimulating the expression of anti-oxidant genes (MnSOD and catalase). This adaptive transcriptional response is mediated by the activation of WNT/ß-catenin and FOXO4 signalling. Our data highlights a new functional synergism between oncogenic EGFR and PIK3CA with WNT/ß-catenin conferring high tolerance to oxidative stress generated by nutrient deprivation.
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Receptores ErbB/metabolismo , Glucosa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , beta Catenina/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Trifosfato/metabolismo , Alelos , Antioxidantes/metabolismo , Proteínas de Ciclo Celular , Muerte Celular , Línea Celular , Fosfatidilinositol 3-Quinasa Clase I , Células Epiteliales/metabolismo , Receptores ErbB/genética , Factores de Transcripción Forkhead , Glutatión/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Glándulas Mamarias Humanas/metabolismo , Mutación , Oxidación-Reducción , Estrés Oxidativo , Fosfatidilinositol 3-Quinasas/genética , Superóxido Dismutasa/metabolismo , Factores de Transcripción/metabolismoRESUMEN
INTRODUCTION: In vitro evidence suggests that PIK3CA (phosphatidylinositol 3-kinase, catalytic, alpha polypeptide) activation may be associated with altered chemotherapy sensitivity in cancer. METHODS: Tumor DNA from 140 patients with stage II-III breast cancer undergoing neoadjuvant chemotherapy was sequenced for PIK3CA mutations on exons 1, 9, and 20. Mutation status was correlated with clinical/pathological parameters and chemotherapy response as (a) pathological complete response (pCR) versus residual cancer or (b) quantitative residual cancer burden (RCB) scores, including stratification for estrogen receptor (ER) expression status, type of chemotherapy, and by exons. RESULTS: Twenty-three patients (16.4%) harbored a PIK3CA mutation, with 12, 11, and 0 mutations located in exons 9, 20, and 1, respectively. PIK3CA exon 9 mutations were more frequent among node-negative (52% versus 25%; P = 0.012) than node-positive tumors, particularly among ER-positive tumors. pCR rates and RCB scores were similar among patients with the wild-type and mutant PIK3CA genes, even after stratification by ER status, chemotherapy regimen (anthracycline versus anthracycline plus paclitaxel), or exon. CONCLUSION: PIK3CA mutations are not associated with altered sensitivity to preoperative anthracycline-based or taxane-based chemotherapies in ER-positive and ER-negative breast tumors. In this study, PIK3CA mutation was associated with a decreased rate of node-positive disease, particularly among ER-positive tumors.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Mutación , Fosfatidilinositol 3-Quinasas/genética , Adulto , Anciano , Antraciclinas/administración & dosificación , Neoplasias de la Mama/química , Neoplasias de la Mama/patología , Quimioterapia Adyuvante , Fosfatidilinositol 3-Quinasa Clase I , Análisis Mutacional de ADN , Femenino , Humanos , Metástasis Linfática , Persona de Mediana Edad , Terapia Neoadyuvante/métodos , Estadificación de Neoplasias , Receptor ErbB-2/análisis , Receptores de Estrógenos/análisis , Receptores de Progesterona/análisis , Taxoides/administración & dosificaciónRESUMEN
The molecular machinery that governs circadian rhythmicity is based on clock proteins organized in regulatory feedback loops. Although posttranslational modification of clock proteins is likely to finely control their circadian functions, only limited information is available to date. Here, we show that BMAL1, an essential transcription factor component of the clock mechanism, is SUMOylated on a highly conserved lysine residue (Lys259) in vivo. BMAL1 shows a circadian pattern of SUMOylation that parallels its activation in the mouse liver. SUMOylation of BMAL1 requires and is induced by CLOCK, the heterodimerization partner of BMAL1. Ectopic expression of a SUMO-deficient BMAL1 demonstrates that SUMOylation plays an important role in BMAL1 circadian expression and clock rhythmicity. This reveals an additional level of regulation within the core mechanism of the circadian clock.
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Ritmo Circadiano , Proteína SUMO-1/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción ARNTL , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas CLOCK , Células COS , Proteínas de Ciclo Celular , Línea Celular , Dimerización , Etilmaleimida/farmacología , Regulación de la Expresión Génica , Hígado/metabolismo , Lisina/metabolismo , Ratones , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , Fosforilación , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
The cell cycle and the circadian clock are endogenous pacemakers, which coexist in most eukaryotic cells and share a number of conceptual features. In the zebrafish, light directly regulates the timing of both clocks, although the signaling and transcriptional pathways that convey photic information to essential nuclear regulators have yet to be deciphered. We have previously established the Z3 cell line, which recapitulates the features of zebrafish circadian clock and represents an ideal system to study light-dependent signaling and gene regulation. We conducted a search for light-responsive transcription factors and found that AP-1 DNA binding is highly induced. Light induces the expression of zWee1, a cell cycle gene essential for G2/M transition, and zCry1a, a clock gene of the feedback regulatory loop. We have found consensus AP-1 sites in the regulatory regions of both zWee1 and zCry1a genes, and we show that light inducibility of both genes is abrogated by inhibition of AP-1 function. Light also elicits chromatin remodeling by stimulating hyperacetylation at Lys-14 of histone H3 at both zWee1 and zCry1a promoters, as assessed by chromatin immunoprecipitation assays by using anti-Fos antibody. These findings provide strong evidence that circadian and cell cycle clocks share unique light-responsive pathways in zebrafish.
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Relojes Biológicos/fisiología , Proteínas de Ciclo Celular/metabolismo , Flavoproteínas/metabolismo , Regulación de la Expresión Génica/fisiología , Luz , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción AP-1/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/fisiología , Animales , Western Blotting , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Línea Celular , Inmunoprecipitación de Cromatina , Ritmo Circadiano/fisiología , Clonación Molecular , Criptocromos , Flavoproteínas/genética , Proteínas Nucleares/genética , Regiones Promotoras Genéticas/genética , Proteínas Tirosina Quinasas/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
The zebrafish probably constitutes the best animal system to study the complexity of the circadian clock machinery and the influence that light has on it. The possibilities of producing transgenic fishes, to establish light-responsive cultured cells, and to directly explore light phototransduction on single clock cells are all remarkable features of this circadian system. This article describes some of the most useful methodologies to analyze the behavioral, cellular, and molecular aspects of the zebrafish circadian clock system.
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Ritmo Circadiano/fisiología , Pez Cebra/fisiología , Animales , Conducta Animal , Técnicas de Cultivo de Célula/métodos , Línea Celular , Ritmo Circadiano/efectos de la radiación , Técnicas Genéticas , Larva/crecimiento & desarrollo , Mediciones Luminiscentes/métodos , Técnicas de Cultivo de Órganos/métodos , Estimulación Luminosa , Transducción de Señal/efectos de la radiación , Transgenes , Pez Cebra/genética , Proteínas de Pez Cebra/aislamiento & purificaciónRESUMEN
PERIOD proteins are central components of the Drosophila and mammalian circadian clock. Their function is controlled by daily changes in synthesis, cellular localization, phosphorylation, degradation, as well as specific interactions with other clock components. Here we present the crystal structure of a Drosophila PERIOD (dPER) fragment comprising two tandemly organized PAS (PER-ARNT-SIM) domains (PAS-A and PAS-B) and two additional C-terminal alpha helices (alphaE and alphaF). Our analysis reveals a noncrystallographic dPER dimer mediated by intermolecular interactions of PAS-A with PAS-B and helix alphaF. We show that alphaF is essential for dPER homodimerization and that the PAS-A-alphaF interaction plays a crucial role in dPER clock function, as it is affected by the 29 hr long-period perL mutation.
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Proteínas de Drosophila/química , Proteínas Nucleares/química , Secuencia de Aminoácidos , Animales , Ritmo Circadiano , Cristalografía por Rayos X , Dimerización , Drosophila/química , Drosophila/genética , Drosophila/fisiología , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Proteínas Circadianas Period , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Secuencias Repetitivas de Aminoácido , Homología de Secuencia de Aminoácido , Electricidad EstáticaRESUMEN
A-kinase anchor protein 121 (AKAP121) and its spliced isoform AKAP84 anchor protein kinase A (PKA) to the outer membrane of mitochondria, focusing and enhancing cyclic AMP signal transduction to the organelle. We find that AKAP121/84 also binds PTPD1, a src-associated protein tyrosine phosphatase. A signaling complex containing AKAP121, PKA, PTPD1, and src is assembled in vivo. PTPD1 activates src tyrosine kinase and increases the magnitude and duration of epidermal growth factor (EGF) signaling. EGF receptor phosphorylation and downstream activation of ERK 1/2 and Elk1-dependent gene transcription are enhanced by PTPD1. Expression of a PTPD1 mutant lacking catalytic activity inhibits src and downregulates ERK 1/2 but does not affect the activity of c-Jun N-terminal kinase 1/2 and p38alpha mitogen-activated protein kinase. AKAP121 binds to and redistributes PTPD1 from the cytoplasm to mitochondria and inhibits EGF signaling. Our findings indicate that PTPD1 is a novel positive regulator of src signaling and a key component of the EGF transduction pathway. By binding and/or targeting the phosphatase on mitochondria, AKAP121 modulates the amplitude and persistence of src-dependent EGF transduction pathway. This represents the first example of physical and functional interaction between AKAPs and a protein tyrosine phosphatase.
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Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Transducción de Señal/fisiología , Familia-src Quinasas/metabolismo , Proteínas de Anclaje a la Quinasa A , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Humanos , Mitocondrias/metabolismo , Fosforilación , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Fosfatasas no ReceptorasRESUMEN
A-Kinase anchor proteins (AKAPs) immobilize and concentrate protein kinase A (PKA) isoforms at specific subcellular compartments. Intracellular targeting of PKA holoenzyme elicits rapid and efficient phosphorylation of target proteins, thereby increasing sensitivity of downstream effectors to cAMP action. AKAP121 targets PKA to the cytoplasmic surface of mitochondria. Here we show that conditional expression of AKAP121 in PC12 cells selectively enhances cAMP.PKA signaling to mitochondria. AKAP121 induction stimulates PKA-dependent phosphorylation of the proapoptotic protein BAD at Ser(155), inhibits release of cytochrome c from mitochondria, and protects cells from apoptosis. An AKAP121 derivative mutant that localizes on mitochondria but does not bind PKA down-regulates PKA signaling to the mitochondria and promotes apoptosis. These findings indicate that PKA anchored by AKAP121 transduces cAMP signals to the mitochondria, and it may play an important role in mitochondrial physiology.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/fisiología , AMP Cíclico/metabolismo , Mitocondrias/metabolismo , Transducción de Señal , Proteínas de Anclaje a la Quinasa A , Animales , Apoptosis , Secuencia de Bases , Caspasas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cartilla de ADN , Activación Enzimática , Mitocondrias/enzimología , Células PC12 , Fosforilación , RatasRESUMEN
Circadian rhythms are regulated by clocks located in specific structures of the CNS, such as the suprachiasmatic nucleus (SCN) in mammals, and by peripheral oscillators present in various other tissues. The expression of essential clock genes oscillates both in the SCN and in peripheral pacemakers. Peripheral tissues in the fly and in the fish are directly photoreceptive. In particular, we have established the Z3 embryonic zebrafish cell line that recapitulates the dynamic light-dependent regulation of the vertebrate clock in vitro. In mammals the synchronization to daily light cycles involves neural connections from a subset of light-sensitive receptor-containing retinal ganglion cells. Humoral and/or hormonal signals originating from the SCN are thought to provide timing cues to peripheral clocks. However, alternative routes exist, as some peripheral clocks in mammals can be specifically entrained in a SCN-independent manner by restricted feeding regimes. Thus, not all peripheral tissues are equal in circadian rhythmicity. Testis, for example, displays no intrinsic circadian rhythmicity and the molecular mechanisms of clock gene activation in male germ cells appear to differ from other tissues. The study of the connecting routes that link the SCN to peripheral tissues is likely to reveal signalling pathways of fundamental physiological significance.