RESUMEN
Investigation of drug-induced liver injuries requires appropriate in vivo and in vitro toxicological model systems. In our study, an attempt was made to compare the hepatocarcinoma HepG2 and the stem cell-derived HepaRG cell lines both in two- and three-dimensional culture conditions to find the most suitable model. Comparison of the liver-specific characteristics of these models was performed via the extent and mechanism of acetaminophen (APAP)-induced hepatotoxicity. Investigating the detailed mechanism of APAP-induced hepatotoxicity, different specific cell death inhibitors were used: the pan-caspase inhibitor zVAD-fmk and dabrafenib significantly protected both cell lines from APAP-induced cell death. However, the known specific inhibitors of necroptosis (necrostatin-1 and MDIVI) were only effective in differentiated HepaRG, which suggest a differential execution of activated pathways in the two models. By applying 3D culture methods, CYP2E1 mRNA levels could be elevated, but we failed to achieve a significant increase in hepatocyte function; hence, the 3D cultivation especially in APAP toxicity studies is not necessarily worth the complicated maintenance. Based on our findings, the hepatocyte functions of HepaRG may stand between the properties of HepG2 cells and primary hepatocytes (PHHs). However, it should be noted that in contrast to PHHs having many limitations, HepaRG cells are relatively immortal, having a stable phenotype and CYP450 expression.
RESUMEN
HSF1 (heat shock factor 1) is an evolutionarily conserved master transcriptional regulator of the heat shock response (HSR) in eukaryotic cells. In response to high temperatures, HSF1 upregulates genes encoding molecular chaperones, also called heat shock proteins, which assist the refolding or degradation of damaged intracellular proteins. Accumulating evidence reveals however that HSF1 participates in several other physiological and pathological processes such as differentiation, immune response, and multidrug resistance, as well as in ageing, neurodegenerative demise, and cancer. To address how HSF1 controls these processes one should systematically analyze its target genes. Here we present a novel database called HSF1Base (hsf1base.org) that contains a nearly comprehensive list of HSF1 target genes identified so far. The list was obtained by manually curating publications on individual HSF1 targets and analyzing relevant high throughput transcriptomic and chromatin immunoprecipitation data derived from the literature and the Yeastract database. To support the biological relevance of HSF1 targets identified by high throughput methods, we performed an enrichment analysis of (potential) HSF1 targets across different tissues/cell types and organisms. We found that general HSF1 functions (targets are expressed in all tissues/cell types) are mostly related to cellular proteostasis. Furthermore, HSF1 targets that are conserved across various animal taxa operate mostly in cellular stress pathways (e.g., autophagy), chromatin remodeling, ribosome biogenesis, and ageing. Together, these data highlight diverse roles for HSF1, expanding far beyond the HSR.
Asunto(s)
Proteínas de Choque Térmico/genética , Animales , Cromatina/genética , Ensamble y Desensamble de Cromatina/genética , Inmunoprecipitación de Cromatina/métodos , Humanos , Ratones , Chaperonas Moleculares/genética , Proteostasis/genética , Factores de Transcripción/genéticaRESUMEN
PURPOSE: Multiple endocrine neoplasia type 1 is a rare tumor syndrome caused by germline mutations of MEN1 gene. Phenotype varies widely, and no definitive correlation with the genotype has been observed. Mutation-negative patients with MEN1-associated tumors represent phenocopies. By comparing mutation-positive and mutation-negative patients, we aimed to identify phenotype features predictive for a positive genetic test and to evaluate the role of MEN1 mutations in phenotype modulation. METHODS: Mutation screeening of MEN1 gene by Sanger sequencing and assessment of clinical data of 189 consecutively enrolled probands and relatives were performed at our national and European Reference Center. Multiple ligation probe amplification analysis of MEN1 gene and Sanger sequencing of CDKN1B were carried out in clinically suspicious but MEN1-negative cases. RESULTS: Twenty-seven probands and twenty family members carried MEN1 mutations. Five mutations have not been described earlier. Pronouncedly high number of phenocopies (>70%) was observed. Clinical suspicion of MEN1 syndrome emerged at significantly earlier age in MEN1-positive compared to MEN1-negative probands. Gastroenteropancreatic neuroendocrine tumors developed significantly earlier and more frequently in carriers compared to non-carriers. Probands with high-impact (frameshift, nonsense, large deletions) mutations, predicted to affect menin function significantly, developed GEP-NETs more frequently compared to low-impact (inframe and missense) mutation carriers. CONCLUSIONS: MEN1 phenocopy is common and represents a significant confounder for the genetic testing. GEP-NET under 30 years best predicted a MEN1 mutation. The present study thus confirmed a previous proposal and suggested that GEP-NET under 30 years should be considered as a part of the indication criteria for MEN1 mutational analysis.
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Neoplasia Endocrina Múltiple Tipo 1/genética , Proteínas Proto-Oncogénicas/genética , Adulto , Edad de Inicio , Anciano , Análisis Mutacional de ADN , Femenino , Estudios de Asociación Genética , Pruebas Genéticas , Humanos , Hungría/epidemiología , Incidencia , Masculino , Persona de Mediana Edad , Neoplasia Endocrina Múltiple Tipo 1/epidemiología , Mutación , Penetrancia , Estudios RetrospectivosRESUMEN
The A-kinase anchoring protein (AKAP) GSK3ß interaction protein (GSKIP) is a cytosolic scaffolding protein binding protein kinase A (PKA) and glycogen synthase kinase 3ß (GSK3ß). Here we show that both the AKAP function of GSKIP, i.e. its direct interaction with PKA, and its direct interaction with GSK3ß are required for the regulation of ß-catenin and thus Wnt signaling. A cytoplasmic destruction complex targets ß-catenin for degradation and thus prevents Wnt signaling. Wnt signals cause ß-catenin accumulation and translocation into the nucleus, where it induces Wnt target gene expression. GSKIP facilitates control of the ß-catenin stabilizing phosphorylation at Ser-675 by PKA. Its interaction with GSK3ß facilitates control of the destabilizing phosphorylation of ß-catenin at Ser-33/Ser-37/Thr-41. The influence of GSKIP on ß-catenin is explained by its scavenger function; it recruits the kinases away from the destruction complex without forming a complex with ß-catenin. The regulation of ß-catenin by GSKIP is specific for this AKAP as AKAP220, which also binds PKA and GSK3ß, did not affect Wnt signaling. We find that the binding domain of AKAP220 for GSK3ß is a conserved GSK3ß interaction domain (GID), which is also present in GSKIP. Our findings highlight an essential compartmentalization of both PKA and GSK3ß by GSKIP, and ascribe a function to a cytosolic AKAP-PKA interaction as a regulatory factor in the control of canonical Wnt signaling. Wnt signaling controls different biological processes, including embryonic development, cell cycle progression, glycogen metabolism, and immune regulation; deregulation is associated with diseases such as cancer, type 2 diabetes, inflammatory, and Alzheimer's and Parkinson's diseases.
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Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Proteínas Represoras/metabolismo , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismo , Proteínas de Anclaje a la Quinasa A , Células A549 , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Glucógeno Sintasa Quinasa 3 beta/genética , Células HEK293 , Células HeLa , Humanos , Dominios Proteicos , Proteínas Represoras/genética , beta Catenina/genéticaRESUMEN
A-kinase anchoring proteins (AKAPs) control the localization of cAMP-dependent protein kinase A (PKA) by tethering PKA to distinct cellular compartments. Through additional direct proteinprotein interactions with PKA substrates and other signaling molecules they form multi-protein complexes. Thereby, AKAPs regulate the access of PKA to its substrates in a temporal and spatial manner as well as the local crosstalk of cAMP/PKA with other signaling pathways. Due to the increasing information on their molecular functioning and three-dimensional structures, and their emerging roles in the development of diseases, AKAPs move into the focus as potential drug targets. Targeting AKAP dependent protein-protein interactions for interference with local signal processing inside cells potentially allows for the development of therapeutics with high selectivity and fewer side effects.
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Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Terapia Molecular Dirigida , Proteínas de Anclaje a la Quinasa A/química , Animales , Diseño de Fármacos , Humanos , Conformación Proteica , Transducción de Señal/efectos de los fármacosRESUMEN
A-kinase anchoring proteins (AKAPs) represent a family of structurally diverse proteins, all of which bind PKA. A member of this family is glycogen synthase kinase 3ß (GSK3ß) interaction protein (GSKIP). GSKIP interacts with PKA and also directly interacts with GSK3ß. The physiological function of the GSKIP protein in vivo is unknown. We developed and characterized a conditional knock-out mouse model and found that GSKIP deficiency caused lethality at birth. Embryos obtained through Caesarean section at embryonic day 18.5 were cyanotic, suffered from respiratory distress, and failed to initiate breathing properly. Additionally, all GSKIP-deficient embryos showed an incomplete closure of the palatal shelves accompanied by a delay in ossification along the fusion area of secondary palatal bones. On the molecular level, GSKIP deficiency resulted in decreased phosphorylation of GSK3ß at Ser-9 starting early in development (embryonic day 10.5), leading to enhanced GSK3ß activity. At embryonic day 18.5, GSK3ß activity decreased to levels close to that of wild type. Our findings reveal a novel, crucial role for GSKIP in the coordination of GSK3ß signaling in palatal shelf fusion.
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Glucógeno Sintasa Quinasa 3/metabolismo , Hueso Paladar/embriología , Hueso Paladar/metabolismo , Proteínas Represoras/metabolismo , Alelos , Animales , Fisura del Paladar/embriología , Fisura del Paladar/enzimología , Fisura del Paladar/patología , Pérdida del Embrión/metabolismo , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/embriología , Embrión de Mamíferos/enzimología , Femenino , Regulación del Desarrollo de la Expresión Génica , Glucógeno Sintasa Quinasa 3 beta , Hemicigoto , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Hueso Paladar/anomalías , Hueso Paladar/enzimología , Fenotipo , Fosforilación , Fosfoserina/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/deficiencia , RespiraciónRESUMEN
The second messenger cyclic adenosine monophosphate (cAMP) can bind and activate protein kinase A (PKA). The cAMP/PKA system is ubiquitous and involved in a wide array of biological processes and therefore requires tight spatial and temporal regulation. Important components of the safeguard system are the A-kinase anchoring proteins (AKAPs), a heterogeneous family of scaffolding proteins defined by its ability to directly bind PKA. AKAPs tether PKA to specific subcellular compartments, and they bind further interaction partners to create local signalling hubs. The recent discovery of new AKAPs and advances in the field that shed light on the relevance of these hubs for human disease highlight unique opportunities for pharmacological modulation. This review exemplifies how interference with signalling, particularly cAMP signalling, at such hubs can reshape signalling responses and discusses how this could lead to novel pharmacological concepts for the treatment of disease with an unmet medical need such as cardiovascular disease and cancer.
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Proteínas de Anclaje a la Quinasa A/fisiología , AMP Cíclico/metabolismo , Sistemas de Mensajero Secundario , Secuencia de Aminoácidos , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Fármacos Cardiovasculares/farmacología , Fármacos Cardiovasculares/uso terapéutico , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/metabolismo , Secuencia Conservada , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Datos de Secuencia Molecular , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Mapas de Interacción de ProteínasRESUMEN
The mitochondrial antioxidant homeostasis was investigated in Arabidopsis ppr40-1 mutant, which presents a block of electron flow at complex III. The activity of the ascorbate biosynthetic enzyme, L-galactono-1,4-lactone dehydrogenase (EC 1.3.2.3) (GLDH) was elevated in mitochondria isolated from mutant plants. In addition increased activities of the enzymes of Foyer-Halliwell-Asada cycle and elevated glutathione (GSH) level were observed in the mutant mitochondria. Lower ascorbate and ascorbate plus dehydroascorbate contents were detected at both cellular and mitochondrial level. Moreover, the more oxidized mitochondrial redox status of ascorbate in the ppr40-1 mutant indicated that neither the enhanced activity of GLDH nor Foyer-Halliwell-Asada cycle could compensate for the enhanced ascorbate consumption in the absence of a functional respiratory chain.
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Arabidopsis/metabolismo , Ácido Ascórbico/metabolismo , Glutatión/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Antioxidantes/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ascorbato Peroxidasas/metabolismo , Ácido Ascórbico/biosíntesis , Respiración de la Célula , Ácido Deshidroascórbico/metabolismo , Complejo III de Transporte de Electrones/genética , Complejo III de Transporte de Electrones/metabolismo , Glutatión Reductasa/metabolismo , Mitocondrias/metabolismo , Mutación , NADH NADPH Oxidorreductasas/metabolismoRESUMEN
Genes encoding the tail proteins of the temperate phage 16-3 of the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti 41 have been identified. First, a new host range gene, designated hII, was localized by using missense mutations. The corresponding protein was shown to be identical to the 85-kDa tail protein by determining its N-terminal sequence. Electron microscopic analysis showed that phage 16-3 possesses an icosahedral head and a long, noncontractile tail characteristic of the Siphoviridae. By using a lysogenic S. meliloti 41 strain, mutants with insertions in the putative tail region of the genome were constructed and virion morphology was examined after induction of the lytic cycle. Insertions in ORF017, ORF018a, ORF020, ORF021, the previously described h gene, and hII resulted in uninfectious head particles lacking tail structures, suggesting that the majority of the genes in this region are essential for tail formation. By using different bacterial mutants, it was also shown that not only the RkpM and RkpY proteins but also the RkpZ protein of the host takes part in the formation of the phage receptor. Results for the host range phage mutants and the receptor mutant bacteria suggest that the HII tail protein interacts with the capsular polysaccharide of the host and that the tail protein encoded by the original h gene recognizes a proteinaceous receptor.
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Bacteriófagos/metabolismo , Regulación Viral de la Expresión Génica/fisiología , Genes Virales/fisiología , Sinorhizobium meliloti/virología , Proteínas de la Cola de los Virus/genética , Proteínas de la Cola de los Virus/metabolismo , Bacteriófagos/genética , Bacteriófagos/ultraestructura , MutaciónRESUMEN
Rhizobial surface polysaccharides, including capsular polysaccharides (KPS), are involved in symbiotic infection. The rkp-3 locus of Sinorhizobium meliloti 41 is responsible for the production of pseudaminic acid, one of the components of the KR5 antigen, a strain-specific KPS. We have extended the sequence determination and genetic dissection of the rkp-3 region to clarify the structure and function of the rkpY gene and to identify additional rkp genes. Except for rkpY, no other genes were found where mutation affected the KPS structure and symbiosis. These mutants show a unique phenotype producing a low molecular weight polysaccharide (LMW PS). Creating double mutants, we have shown that biosynthesis genes of the KR5 antigen except rkpZ are not necessary for the production of this LMW PS. Polysaccharide analysis of genetically modified strains suggests that rkpY has pleiotropic effects on polysaccharide production. It directs KPS synthesis to the KR5 antigen and influences lipo-oligo 3-deoxy-d-manno-2 octulosonic acid (Kdo) production in S. meliloti 41. In addition, rkpY suppresses the lipo-oligoKdo production when it is introduced into S. meliloti 1021.
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Antígenos Bacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Polisacáridos Bacterianos/biosíntesis , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismo , Proteínas Bacterianas/genética , Perfilación de la Expresión Génica , Datos de Secuencia MolecularRESUMEN
The strain-specific capsular polysaccharide KR5 antigen of Sinorhizobium meliloti 41 is required both for invasion of the symbiotic nodule and for the adsorption of bacteriophage 16-3. In order to know more about the genes involved in these events, bacterial mutants carrying an altered phage receptor were identified by using host range phage mutants. A representative mutation was localized in the rkpM gene by complementation and DNA sequence analysis. A host range phage mutant isolated on these phage-resistant bacteria was used to identify the h gene, which is likely to encode the tail fiber protein of phage 16-3. The nucleotide sequences of the h gene as well as a host range mutant allele were also established. In both the bacterial and phage mutant alleles, a missense mutation was found, indicating a direct contact between the RkpM and H proteins in the course of phage adsorption. Some mutations could not be localized in these genes, suggesting that additional components are also important for bacteriophage receptor recognition.