RESUMEN
Heart failure (HF) is the leading cause of death in the Western world. Pathophysiological processes underlying HF development, including cardiac hypertrophy, fibrosis and inflammation, are controlled by specific microRNAs (miRNAs). Whereas most studies investigate miRNA function in one particular cardiac cell type, their multicellular function is poorly investigated. The present study probed 194 miRNAs -differentially expressed in cardiac inflammatory disease - for regulating cardiomyocyte size, cardiac fibroblasts collagen content, and macrophage polarization. Of the tested miRNAs, 13%, 26%, and 41% modulated cardiomyocyte size, fibroblast collagen production, and macrophage polarization, respectively. Seventeen miRNAs affected all three cellular processes, including miRNAs with established (miR-210) and unknown roles in cardiac pathophysiology (miR-145-3p). These miRNAs with a multi-cellular function commonly target various genes. In-depth analysis in vitro of previously unstudied miRNAs revealed that the observed phenotypical alterations concurred with changes in transcript and protein levels of hypertrophy-, fibrosis- and inflammation-related genes. MiR-145-3p and miR-891a-3p were identified to regulate the fibrotic response, whereas miR-223-3p, miR-486-3p, and miR-488-5p modulated macrophage activation and polarisation. In conclusion, miRNAs are multi-cellular regulators of different cellular processes underlying cardiac disease. We identified previously undescribed roles of miRNAs in hypertrophy, fibrosis, and inflammation, and attribute new cellular effects to various well-known miRNAs.
Asunto(s)
Cardiomegalia/patología , Insuficiencia Cardíaca/genética , MicroARNs/metabolismo , Miocarditis/inmunología , Miocardio/patología , Animales , Animales Recién Nacidos , Cardiomegalia/genética , Cardiomegalia/inmunología , Células Cultivadas , Fibroblastos , Fibrosis , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Insuficiencia Cardíaca/inmunología , Insuficiencia Cardíaca/patología , Humanos , Activación de Macrófagos/genética , Activación de Macrófagos/inmunología , Macrófagos , Ratones , Miocarditis/genética , Miocarditis/patología , Miocardio/citología , Miocardio/inmunología , Miocitos Cardíacos , Cultivo Primario de Células , RatasRESUMEN
Chronic Obstructive Pulmonary Disease (COPD) is a highly prevalent condition characterized by inflammation and progressive obstruction of the airways. At present, there is no treatment that suppresses the chronic inflammation of the disease, and COPD patients often succumb to the condition. Excessive oxidative stress caused by smoke inhalation is a major driving force of the disease. The transcription factor NRF2 is a critical player in the battle against oxidative stress and its function is impaired in COPD. Increasing NRF2 activity may therefore be a viable therapeutic option for COPD treatment. We show that down regulation of KEAP1, a NRF2 inhibitor, protects primary human lung epithelial cells from cigarette-smoke-extract (CSE) induced cell death in an established in vitro model of radical stress. To identify new potential drug targets with a similar effect, we performed a siRNA screen of the 'druggable' genome using a NRF2 transcriptional reporter cell line. This screen identified multiple genes that when down regulated increased NRF2 transcriptional activity and provided a survival benefit in the in vitro model. Our results suggest that inhibiting components of the ubiquitin-proteasome system will have the strongest effects on NRF2 transcriptional activity by increasing NRF2 levels. We also find that down regulation of the small GTPase Rab28 or the Estrogen Receptor ESRRA provide a survival benefit. Rab28 knockdown increased NRF2 protein levels, indicating that Rab28 may regulate NRF2 proteolysis. Conversely ESRRA down regulation increased NRF2 transcriptional activity without affecting NRF2 levels, suggesting a proteasome-independent mechanism.
Asunto(s)
Bronquios/citología , Regulación de la Expresión Génica , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo , Mucosa Respiratoria/citología , Fumar/efectos adversos , Bronquios/metabolismo , Muerte Celular , Supervivencia Celular , Células Cultivadas , Descubrimiento de Drogas , Células HEK293 , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Terapia Molecular Dirigida , NAD(P)H Deshidrogenasa (Quinona)/genética , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/genética , Interferencia de ARN , Mucosa Respiratoria/metabolismo , Regulación hacia ArribaRESUMEN
Huntington's disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified new modifiers of mutant HTT toxicity by performing a large-scale 'druggable genome' siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen and also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone αB-crystallin and reduced the aggregation of diverse proteins. We generated new QPCT inhibitors using in silico methods followed by in vitro screening, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a new HD druggable target affecting mutant HTT aggregation and provide proof of principle for a discovery pipeline from druggable genome screen to drug development.
Asunto(s)
Aminoaciltransferasas/efectos de los fármacos , Aminoaciltransferasas/genética , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/genética , ARN Interferente Pequeño , Aminoaciltransferasas/antagonistas & inhibidores , Animales , Células Cultivadas , Biología Computacional , Drosophila , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteína Huntingtina , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Pez Cebra , Cadena B de alfa-Cristalina/metabolismoRESUMEN
The identification of diagnostic markers and therapeutic candidate genes in common diseases is complicated by the involvement of thousands of genes. We hypothesized that genes co-regulated with a key gene in allergy, IL13, would form a module that could help to identify candidate genes. We identified a T helper 2 (TH2) cell module by small interfering RNA-mediated knockdown of 25 putative IL13-regulating transcription factors followed by expression profiling. The module contained candidate genes whose diagnostic potential was supported by clinical studies. Functional studies of human TH2 cells as well as mouse models of allergy showed that deletion of one of the genes, S100A4, resulted in decreased signs of allergy including TH2 cell activation, humoral immunity, and infiltration of effector cells. Specifically, dendritic cells required S100A4 for activating T cells. Treatment with an anti-S100A4 antibody resulted in decreased signs of allergy in the mouse model as well as in allergen-challenged T cells from allergic patients. This strategy, which may be generally applicable to complex diseases, identified and validated an important diagnostic and therapeutic candidate gene in allergy.
Asunto(s)
Estudios de Asociación Genética , Hipersensibilidad/genética , Hipersensibilidad/inmunología , Proteínas S100/genética , Investigación Biomédica Traslacional , Adulto , Animales , Anticuerpos Bloqueadores/farmacología , Polaridad Celular , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Dermatitis/diagnóstico , Dermatitis/genética , Dermatitis/inmunología , Dermatitis/prevención & control , Modelos Animales de Enfermedad , Epítopos/efectos de los fármacos , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Hipersensibilidad/diagnóstico , Hipersensibilidad/prevención & control , Memoria Inmunológica/efectos de los fármacos , Interleucina-13/metabolismo , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Modelos Inmunológicos , Rinitis Alérgica Estacional/diagnóstico , Rinitis Alérgica Estacional/genética , Rinitis Alérgica Estacional/inmunología , Rinitis Alérgica Estacional/patología , Proteína de Unión al Calcio S100A4 , Proteínas S100/deficiencia , Células Th2/citología , Células Th2/inmunologíaRESUMEN
The Th2 cytokine IL-13 plays a key role in allergy, by regulating IgE, airway hyper secretion, eosinophils and mast cells. In this study, we aimed to identify novel transcription factors (TFs) that potentially regulated IL-13. We analyzed Th2 polarized naïve T cells from four different blood donors with gene expression microarrays to find clusters of genes that were correlated or anti-correlated with IL13. These clusters were further filtered, by selecting genes that were functionally related. In these clusters, we identified three transcription factors (TFs) that were predicted to regulate the expression of IL13, namely CEBPB, E2F6 and AHR. siRNA mediated knockdowns of these TFs in naïve polarized T cells showed significant increases of IL13, following knockdown of CEBPB and E2F6, but not AHR. This suggested an inhibitory role of CEBPB and E2F6 in the regulation of IL13 and allergy. This was supported by analysis of E2F6, but not CEBPB, in allergen-challenged CD4+ T cells from six allergic patients and six healthy controls, which showed decreased expression of E2F6 in patients. In summary, our findings indicate an inhibitory role of E2F6 in the regulation of IL-13 and allergy. The analytical approach may be generally applicable to elucidate the complex regulatory patterns in Th2 cell polarization and allergy.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Factor de Transcripción E2F6/metabolismo , Interleucina-13/metabolismo , Rinitis Alérgica Estacional/metabolismo , Adulto , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteína beta Potenciadora de Unión a CCAAT/genética , Linfocitos T CD4-Positivos/metabolismo , Análisis por Conglomerados , Factor de Transcripción E2F6/genética , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Interleucina-5/análisis , Activación de Linfocitos , Masculino , Interferencia de ARN , ARN Interferente Pequeño , Receptores de Hidrocarburo de Aril/genética , Rinitis Alérgica Estacional/genética , Rinitis Alérgica Estacional/inmunología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Plasmodium parasites undergo a clinically silent and obligatory developmental phase in the host's liver cells before they are able to infect erythrocytes and cause malaria symptoms. To overcome the scarcity of compounds targeting the liver stage of malaria, we screened a library of 1037 existing drugs for their ability to inhibit Plasmodium hepatic development. Decoquinate emerged as the strongest inhibitor of Plasmodium liver stages, both in vitro and in vivo. Furthermore, decoquinate kills the parasite's replicative blood stages and is active against developing gametocytes, the forms responsible for transmission. The drug acts by selectively and specifically inhibiting the parasite's mitochondrial bc(1) complex, with little cross-resistance with the antimalarial drug atovaquone. Oral administration of a single dose of decoquinate effectively prevents the appearance of disease, warranting its exploitation as a potent antimalarial compound.
Asunto(s)
Antimaláricos/farmacología , Hepatocitos/parasitología , Malaria/tratamiento farmacológico , Malaria/parasitología , Plasmodium/efectos de los fármacos , Animales , Atovacuona/farmacología , Línea Celular Tumoral , Decoquinato/farmacología , Evaluación Preclínica de Medicamentos/métodos , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Moleculares , Estructura Molecular , Conformación ProteicaRESUMEN
RNA interference (RNAi) has become an important technique for loss-of-gene-function studies in mammalian cells. To achieve reliable results in an RNAi experiment, efficient and specific silencing triggers are required. Here we present genome-wide data sets for the production of endoribonuclease-prepared short interfering RNAs (esiRNAs) for human, mouse and rat. We used an algorithm to predict the optimal region for esiRNA synthesis for every protein-coding gene of these three species. We created a database, RiDDLE, for retrieval of target sequences and primer information. To test this in silico resource experimentally, we generated 16,242 esiRNAs that can be used for RNAi screening in human cells. Comparative analyses with chemically synthesized siRNAs demonstrated a high silencing efficacy of esiRNAs and a 12-fold reduction of downregulated off-target transcripts as detected by microarray analysis. Hence, the presented esiRNA libraries offer an efficient, cost-effective and specific alternative to presently available mammalian RNAi resources.
Asunto(s)
Endorribonucleasas/genética , Biblioteca Genómica , Genómica/métodos , Interferencia de ARN , ARN Interferente Pequeño/genética , Animales , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Transcripción Genética , Transfección , Regiones no Traducidas , Interfaz Usuario-ComputadorRESUMEN
Although numerous fundamental aspects of development have been uncovered through the study of individual genes and proteins, system-level models are still missing for most developmental processes. The first two cell divisions of Caenorhabditis elegans embryogenesis constitute an ideal test bed for a system-level approach. Early embryogenesis, including processes such as cell division and establishment of cellular polarity, is readily amenable to large-scale functional analysis. A first step toward a system-level understanding is to provide 'first-draft' models both of the molecular assemblies involved and of the functional connections between them. Here we show that such models can be derived from an integrated gene/protein network generated from three different types of functional relationship: protein interaction, expression profiling similarity and phenotypic profiling similarity, as estimated from detailed early embryonic RNA interference phenotypes systematically recorded for hundreds of early embryogenesis genes. The topology of the integrated network suggests that C. elegans early embryogenesis is achieved through coordination of a limited set of molecular machines. We assessed the overall predictive value of such molecular machine models by dynamic localization of ten previously uncharacterized proteins within the living embryo.
Asunto(s)
Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Desarrollo Embrionario , Modelos Biológicos , Biología de Sistemas/métodos , Algoritmos , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , División Celular , Polaridad Celular , Desarrollo Embrionario/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Fenotipo , Unión Proteica , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
During the past five years, RNA interference (RNAi) has emerged as arguably the best functional genomics tool available to date, providing direct, causal links between individual genes and loss-of-function phenotypes through robust, broadly applicable, and readily upscalable methodologies. Originally applied experimentally in C. elegans and Drosophila, RNAi is now widely used in mammalian cell systems also. The development of commercially available libraries of short interfering RNAs (siRNAs) and other RNAi silencing reagents targeting entire classes of human genes provide the opportunity to carry out genome-scale screens to discover and characterize gene functions directly in human cells. A key challenge of these studies, also faced by earlier genomics or proteomics approaches, resides in reaching an optimal balance between the necessarily high throughput and the desire to achieve the same level of detailed analysis that is routine in conventional small-scale studies. This chapter discusses technical aspects of how to perform such screens, what parameters to monitor, and which readouts to apply. Examples of homogenous assays and multiplexed high-content microscopy-based screens are demonstrated.
Asunto(s)
Genómica , Interferencia de ARN , Apoptosis , Automatización , División Celular , Línea Celular Tumoral , Genes Reporteros , Humanos , ARN Interferente Pequeño/genéticaRESUMEN
The three GTPases Rab5, Rab4 and Rab11 regulate sequential transport steps along the endocytic/recycling pathway, and occupy distinct membrane domains on early and recycling endosomes. To address the mechanisms that regulate communication between such domains, we searched for proteins that interact with both Rab5 and Rab4. Here, we report that Rabenosyn-5, a previously identified Rab5 effector, also binds to Rab4. Rabenosyn-5 overexpression increased the association between Rab5 and Rab4 endosomal domains and decreased the fraction of Rab4- and Rab11-positive structures. This redistribution was accompanied by a faster rate of transferrin recycling from early endosomes to the cell surface and reduced transport to Rab11-containing perinuclear recycling endosomes. These effects depend on the ability of Rabenosyn-5 to interact with Rab4. We propose that divalent Rab effectors regulate protein sorting and recycling by connecting Rab domains on early endosomes.