RESUMEN
Accessing the natural genetic diversity of species unveils hidden genetic traits, clarifies gene functions and allows the generalizability of laboratory findings to be assessed. One notable discovery made in natural isolates of Saccharomyces cerevisiae is that aneuploidy-an imbalance in chromosome copy numbers-is frequent1,2 (around 20%), which seems to contradict the substantial fitness costs and transient nature of aneuploidy when it is engineered in the laboratory3-5. Here we generate a proteomic resource and merge it with genomic1 and transcriptomic6 data for 796 euploid and aneuploid natural isolates. We find that natural and lab-generated aneuploids differ specifically at the proteome. In lab-generated aneuploids, some proteins-especially subunits of protein complexes-show reduced expression, but the overall protein levels correspond to the aneuploid gene dosage. By contrast, in natural isolates, more than 70% of proteins encoded on aneuploid chromosomes are dosage compensated, and average protein levels are shifted towards the euploid state chromosome-wide. At the molecular level, we detect an induction of structural components of the proteasome, increased levels of ubiquitination, and reveal an interdependency of protein turnover rates and attenuation. Our study thus highlights the role of protein turnover in mediating aneuploidy tolerance, and shows the utility of exploiting the natural diversity of species to attain generalizable molecular insights into complex biological processes.
Asunto(s)
Aneuploidia , Complejo de la Endopetidasa Proteasomal , Proteolisis , Proteoma , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Compensación de Dosificación (Genética) , Variación Genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Proteoma/metabolismo , Proteoma/genética , Proteómica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitinación , Perfilación de la Expresión Génica , GenómicaRESUMEN
The assimilation, incorporation, and metabolism of sulfur is a fundamental process across all domains of life, yet how cells deal with varying sulfur availability is not well understood. We studied an unresolved conundrum of sulfur fixation in yeast, in which organosulfur auxotrophy caused by deletion of the homocysteine synthase Met17p is overcome when cells are inoculated at high cell density. In combining the use of self-establishing metabolically cooperating (SeMeCo) communities with proteomic, genetic, and biochemical approaches, we discovered an uncharacterized gene product YLL058Wp, herein named Hydrogen Sulfide Utilizing-1 (HSU1). Hsu1p acts as a homocysteine synthase and allows the cells to substitute for Met17p by reassimilating hydrosulfide ions leaked from met17Δ cells into O-acetyl-homoserine and forming homocysteine. Our results show that cells can cooperate to achieve sulfur fixation, indicating that the collective properties of microbial communities facilitate their basic metabolic capacity to overcome sulfur limitation.
Asunto(s)
Cisteína Sintasa , Metionina , Saccharomyces cerevisiae , Cisteína/metabolismo , Cisteína Sintasa/genética , Cisteína Sintasa/metabolismo , Metionina/metabolismo , Proteómica , Racemetionina , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Azufre/metabolismoRESUMEN
The primary function of the small intestine (SI) is to absorb nutrients to maintain whole-body energy homeostasis. Enterocytes are the major epithelial cell type facilitating nutrient sensing and uptake. However, the molecular regulators governing enterocytes have remained undefined. Here, we identify c-Maf as an enterocyte-specific transcription factor within the SI epithelium. c-Maf expression was determined by opposing Noggin/BMP signals and overlapped with the zonated enrichment of nutrient transporters in the mid-villus region. Functionally, enterocytes required c-Maf to appropriately differentiate along the villus axis. Specifically, gene programs controlling carbohydrate and protein absorption were c-Maf-dependent. Consequently, epithelial cell-specific c-Maf deletion resulted in impaired enterocyte maturation and nutrient uptake, including defects in the adaptation to different nutrient availability. Concomitantly, intraepithelial lymphocytes were less abundant, while commensal epithelial cell-attaching SFB overgrew in a c-Maf-deficient environment, highlighting the close interdependence between the intestinal epithelium, immune system, and microbiota. Collectively, our data identified c-Maf as a key regulator of SI enterocyte differentiation and function, essential for nutrient, immune, and microbial homeostasis.
Asunto(s)
Enterocitos , Intestinos , Proteínas Proto-Oncogénicas c-maf , Animales , Carbohidratos , Enterocitos/metabolismo , Ratones , Nutrientes , Proteínas Proto-Oncogénicas c-maf/genética , Proteínas Proto-Oncogénicas c-maf/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Eukaryotic cells can survive the loss of their mitochondrial genome, but consequently suffer from severe growth defects. 'Petite yeasts', characterized by mitochondrial genome loss, are instrumental for studying mitochondrial function and physiology. However, the molecular cause of their reduced growth rate remains an open question. Here we show that petite cells suffer from an insufficient capacity to synthesize glutamate, glutamine, leucine and arginine, negatively impacting their growth. Using a combination of molecular genetics and omics approaches, we demonstrate the evolution of fast growth overcomes these amino acid deficiencies, by alleviating a perturbation in mitochondrial iron metabolism and by restoring a defect in the mitochondrial tricarboxylic acid cycle, caused by aconitase inhibition. Our results hence explain the slow growth of mitochondrial genome-deficient cells with a partial auxotrophy in four amino acids that results from distorted iron metabolism and an inhibited tricarboxylic acid cycle.
Asunto(s)
Metabolismo Energético , Genoma Mitocondrial , Mitocondrias/genética , Mitocondrias/metabolismo , Levaduras/genética , Levaduras/metabolismo , Aminoácidos/metabolismo , Biomasa , Proliferación Celular , Ciclo del Ácido Cítrico , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Potencial de la Membrana Mitocondrial , Mutación , Fenotipo , Relación Estructura-ActividadRESUMEN
Ubiquitination is a posttranslational modification that regulates many cellular processes including protein degradation, intracellular trafficking, cell signaling, and protein-protein interactions. Deubiquitinating enzymes (DUBs), which reverse the process of ubiquitination, are important regulators of the ubiquitin system. OTUD6B encodes a member of the ovarian tumor domain (OTU)-containing subfamily of deubiquitinating enzymes. Herein, we report biallelic pathogenic variants in OTUD6B in 12 individuals from 6 independent families with an intellectual disability syndrome associated with seizures and dysmorphic features. In subjects with predicted loss-of-function alleles, additional features include global developmental delay, microcephaly, absent speech, hypotonia, growth retardation with prenatal onset, feeding difficulties, structural brain abnormalities, congenital malformations including congenital heart disease, and musculoskeletal features. Homozygous Otud6b knockout mice were subviable, smaller in size, and had congenital heart defects, consistent with the severity of loss-of-function variants in humans. Analysis of peripheral blood mononuclear cells from an affected subject showed reduced incorporation of 19S subunits into 26S proteasomes, decreased chymotrypsin-like activity, and accumulation of ubiquitin-protein conjugates. Our findings suggest a role for OTUD6B in proteasome function, establish that defective OTUD6B function underlies a multisystemic human disorder, and provide additional evidence for the emerging relationship between the ubiquitin system and human disease.
Asunto(s)
Anomalías Múltiples/genética , Endopeptidasas/genética , Discapacidad Intelectual/genética , Adolescente , Animales , Niño , Preescolar , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Humanos , Masculino , Ratones , Linaje , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Convulsiones/genéticaRESUMEN
Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological process in the development of eukaryotic organisms. Dysregulation of this mechanism leads to numerous human neurodegenerative or neurodevelopmental disorders. Through a multi-center collaboration, we identified six de novo genomic deletions and four de novo point mutations involving PSMD12, encoding the non-ATPase subunit PSMD12 (aka RPN5) of the 19S regulator of 26S proteasome complex, in unrelated individuals with intellectual disability, congenital malformations, ophthalmologic anomalies, feeding difficulties, deafness, and subtle dysmorphic facial features. We observed reduced PSMD12 levels and an accumulation of ubiquitinated proteins without any impairment of proteasome catalytic activity. Our PSMD12 loss-of-function zebrafish CRISPR/Cas9 model exhibited microcephaly, decreased convolution of the renal tubules, and abnormal craniofacial morphology. Our data support the biological importance of PSMD12 as a scaffolding subunit in proteasome function during development and neurogenesis in particular; they enable the definition of a neurodevelopmental disorder due to PSMD12 variants, expanding the phenotypic spectrum of UPS-dependent disorders.
Asunto(s)
Trastornos del Neurodesarrollo/genética , Complejo de la Endopetidasa Proteasomal/genética , Adolescente , Animales , Niño , Preescolar , Variaciones en el Número de Copia de ADN , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Eliminación de Gen , Humanos , Lactante , Discapacidad Intelectual/genética , Masculino , Microcefalia/genética , Polimorfismo de Nucleótido Simple , Pez Cebra/genéticaRESUMEN
The supply of MHC class I-restricted peptides is primarily ensured by the degradation of intracellular proteins via the ubiquitin-proteasome system. Depending on the target and the enzymes involved, ubiquitination is a process that may dramatically vary in terms of linkages, length, and attachment sites. Here we identified the unique lysine residue at position 124 of the NY-ESO-1 cancer/testis antigen as the acceptor site for the formation of canonical Lys-48-linkages. Interestingly, a lysine-less form of NY-ESO-1 was as efficient as its wild-type counterpart in supplying the HLA-A*0201-restricted NY-ESO-1157-165 antigenic peptide. In fact, we show that the regulation of NY-ESO-1 processing by the ubiquitin receptors Rpn10 and Rpn13 as a well as by the standard and immunoproteasome is governed by non-canonical ubiquitination on non-lysine sites. In summary, our data underscore the significance of atypical ubiquitination in the modulation of MHC class I antigen processing.
Asunto(s)
Presentación de Antígeno , Antígenos de Neoplasias/inmunología , Antígeno HLA-A2/inmunología , Glicoproteínas de Membrana/inmunología , Proteínas de la Membrana/inmunología , Complejo de la Endopetidasa Proteasomal/inmunología , Ubiquitinación/inmunología , Antígenos de Neoplasias/genética , Antígeno HLA-A2/genética , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Complejo de la Endopetidasa Proteasomal/genética , Proteínas de Unión al ARN , Ubiquitinación/genéticaRESUMEN
Proteasome activator PA28γ (REGγ, Ki antigen) has recently been demonstrated to display anti-apoptotic properties via enhancing Mdm2-p53 interaction, thereby facilitating ubiquitination and down-regulation of the tumor suppressor p53. In this study we demonstrate a correlation between cellular PA28γ levels and the sensitivity of cells towards apoptosis in different cellular contexts thereby confirming a role of proteasome activator PA28γ as an anti-apoptotic regulator. We investigated the anti-apoptotic role of PA28γ upon UV-C stimulation in B8 mouse fibroblasts stably overexpressing the PA28γ-encoding PSME3 gene and upon butyrate-induced apoptosis in human HT29 adenocarcinoma cells with silenced PSME3 gene. Interestingly, our results demonstrate that PA28γ has a strong influence on different apoptotic hallmarks, especially p53 phosphorylation and caspase activation. In detail, PA28γ and effector caspases mutually restrict each other. PA28γ is a caspase substrate, if PA28γ levels are low. In contrast, PA28γ overexpression reduces caspase activities, including the caspase-dependent processing of PA28γ. Furthermore, overexpression of PA28γ resulted in a nuclear accumulation of transcriptional active p53. In summary, our findings indicate that even in a p53-dominated cellular context, pro-apoptotic signaling might be overcome by PA28γ-mediated caspase inhibition.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Autoantígenos/metabolismo , Caspasas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Apoptosis/efectos de la radiación , Butiratos/farmacología , Citocromos c/metabolismo , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de la radiación , Células HT29/efectos de la radiación , Humanos , Ratones Endogámicos BALB C , Mitocondrias/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina/metabolismo , Rayos Ultravioleta/efectos adversosRESUMEN
The metal cadmium (Cd) is a widespread environmental pollutant with documented adverse effects on the kidneys and bones from long-term environmental exposure, but with insufficiently elucidated public health consequences such as risk of cardiovascular disease, hormone-related cancer in adults and developmental effects in children. This study is the first pan-European human biomonitoring project that succeeded in performing harmonized measurements of Cd in urine in a comparable way in mother-child couples from 16 European countries. The aim of the study was to evaluate the overall Cd exposure and significant determinants of Cd exposure. A study population of 1632 women (24-52 years of age), and 1689 children (5-12 years of age), from 32 rural and urban areas, was examined within a core period of 6 months in 2011-2012. Women were stratified as smokers and non-smokers. As expected, smoking mothers had higher geometric mean (gm) urinary cadmium (UCd; 0.24 µg/g crea; n=360) than non-smoking mothers (gm 0.18 µg/g crea; n=1272; p<0.0001), and children had lower UCd (gm 0.065 µg/g crea; n=1689) than their mothers at the country level. Non-smoking women exposed to environmental tobacco smoke (ETS) at home had 14% (95% CI 1-28%) higher UCd than those who were not exposed to ETS at home (p=0.04). No influence of ETS at home or other places on UCd levels was detected in children. Smoking women with primary education as the highest educational level of the household had 48% (95% CI 18-86%) higher UCd than those with tertiary education (p=0.0008). The same observation was seen in non-smoking women and in children; however they were not statistically significant. In children, living in a rural area was associated with 7% (95% CI 1-13%) higher UCd (p=0.03) compared to living in an urban area. Children, 9-12 years had 7% (95% CI 1-13%) higher UCd (p=0.04) than children 5-8 years. About 1% of the mothers, and 0.06% of the children, exceeded the tolerable weekly intake (TWI) appointed by EFSA, corresponding to 1.0 µg Cd/g crea in urine. Poland had the highest UCd in comparison between the 16 countries, while Denmark had the lowest. Whether the differences between countries are related to differences in the degree of environmental Cd contamination or to differences in lifestyle, socioeconomic status or dietary patterns is not clear.
Asunto(s)
Cadmio/orina , Exposición a Riesgos Ambientales/análisis , Contaminantes Ambientales/orina , Adulto , Niño , Preescolar , Estudios Transversales , Monitoreo del Ambiente/métodos , Europa (Continente) , Humanos , Estilo de Vida , Límite de Detección , Persona de Mediana Edad , Madres , Análisis de Regresión , Fumar/metabolismo , Factores Socioeconómicos , Adulto JovenRESUMEN
Within the European Environment and Health Action Plan an initiative to establish a coherent human biomonitoring approach in Europe was started. The project COPHES (COnsortium to Perform Human biomonitoring on a European Scale ) developed recommendations for a harmonized conduct of a human biomonitoring (HBM) survey which came into action as the pilot study DEMOCOPHES (DEMOnstration of a study to COordinate and Perform Human biomonitoring on a European Scale). Seventeen European countries conducted a survey with harmonized instruments for, inter alia, recruitment, fieldwork and sampling, in autumn/winter 2011/2012. Based on the countries' experiences of conducting the pilot study, following lessons learnt were compiled: the harmonized fieldwork instruments (basic questionnaire, urine and hair sampling) turned out to be very valuable for future HBM surveys on the European scale. A school approach was favoured by most of the countries to recruit school-aged children according to the established guidelines and country specific experiences. To avoid a low participation rate, intensive communication with the involved institutions and possible participants proved to be necessary. The communication material should also include information on exclusion criteria and offered incentives. Telephone contact to the participants the day before fieldwork during the survey can prevent the forgetting of appointments and first morning urine samples. To achieve comparable results on the European scale, training of interviewers in all issues of recruitment, fieldwork and sampling through information material and training sessions is crucial. A survey involving many European countries needs time for preparation and conduct. Materials for quality control prepared for all steps of recruitment, fieldwork and sampling proved to be important to warrant reliable results.
Asunto(s)
Salud Ambiental/métodos , Salud Ambiental/organización & administración , Monitoreo del Ambiente/métodos , Cooperación Internacional , Desarrollo de Programa , Proyectos de Investigación/normas , Procesamiento Automatizado de Datos , Salud Ambiental/normas , Monitoreo del Ambiente/normas , Europa (Continente) , Guías como Asunto , Personal de Salud/normas , Humanos , Consentimiento Informado , Relaciones Interprofesionales , Proyectos Piloto , Control de Calidad , Proyectos de Investigación/legislación & jurisprudencia , Muestreo , Encuestas y Cuestionarios/normasRESUMEN
BACKGROUND: For Europe as a whole, data on internal exposure to environmental chemicals do not yet exist. Characterization of the internal individual chemical environment is expected to enhance understanding of the environmental threats to health. OBJECTIVES: We developed and applied a harmonized protocol to collect comparable human biomonitoring data all over Europe. METHODS: In 17 European countries, we measured mercury in hair and cotinine, phthalate metabolites, and cadmium in urine of 1,844 children (5-11 years of age) and their mothers. Specimens were collected over a 5-month period in 2011-2012. We obtained information on personal characteristics, environment, and lifestyle. We used the resulting database to compare concentrations of exposure biomarkers within Europe, to identify determinants of exposure, and to compare exposure biomarkers with health-based guidelines. RESULTS: Biomarker concentrations showed a wide variability in the European population. However, levels in children and mothers were highly correlated. Most biomarker concentrations were below the health-based guidance values. CONCLUSIONS: We have taken the first steps to assess personal chemical exposures in Europe as a whole. Key success factors were the harmonized protocol development, intensive training and capacity building for field work, chemical analysis and communication, as well as stringent quality control programs for chemical and data analysis. Our project demonstrates the feasibility of a Europe-wide human biomonitoring framework to support the decision-making process of environmental measures to protect public health.
Asunto(s)
Cotinina/orina , Exposición a Riesgos Ambientales/estadística & datos numéricos , Monitoreo del Ambiente/métodos , Contaminantes Ambientales/metabolismo , Adulto , Biomarcadores/orina , Cadmio/orina , Niño , Preescolar , Exposición a Riesgos Ambientales/análisis , Contaminantes Ambientales/análisis , Contaminantes Ambientales/orina , Europa (Continente)/epidemiología , Femenino , Cabello/química , Humanos , Masculino , Mercurio/análisis , Madres , Ácidos Ftálicos/orinaRESUMEN
Antitumor vaccination using synthetic long peptides (SLP) is an additional therapeutic strategy currently under development. It aims to activate tumor-specific CD8(+) CTL by professional APCs such as DCs. DCs can activate T lymphocytes by MHC class I presentation of exogenous antigens - a process referred to as "cross-presentation". Until recently, the intracellular mechanisms involved in cross-presentation of soluble antigens have been unclear. Here, we characterize the cross-presentation pathway of SLP Melan-A16-40 containing the HLA-A2-restricted epitope26-35 (A27L) in human DCs. Using confocal microscopy and specific inhibitors, we show that SLP16-40 is rapidly taken up by DC and follows a classical TAP- and proteasome-dependent cross-presentation pathway. Our data support a role for the ER-associated degradation machinery (ERAD)-related protein p97/VCP in the transport of SLP16-40 from early endosomes to the cytoplasm but formally exclude both sec61 and Derlin-1 as possible retro-translocation channels for cross-presentation. In addition, we show that generation of the Melan-A26-35 peptide from the SLP16-40 was absolutely not influenced by the proteasome subunit composition in DC. Altogether, our findings propose a model for cross-presentation of SLP which tends to enlarge the repertoire of potential candidates for retro-translocation of exogenous antigens to the cytosol.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos/inmunología , Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Péptidos/inmunología , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/metabolismo , Antígenos/química , Antígenos de Neoplasias/química , Antígenos de Neoplasias/inmunología , Proteínas de Ciclo Celular/metabolismo , Células Dendríticas/metabolismo , Endocitosis/inmunología , Degradación Asociada con el Retículo Endoplásmico , Endosomas/metabolismo , Humanos , Cinética , Lisosomas/metabolismo , Antígeno MART-1/química , Antígeno MART-1/inmunología , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Péptidos/síntesis química , Péptidos/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Transporte de Proteínas , Canales de Translocación SEC , Proteína que Contiene ValosinaRESUMEN
COPHES/DEMOCOPHES has its origins in the European Environment and Health Action Plan of 2004 to "develop a coherent approach on human biomonitoring (HBM) in Europe". Within this twin-project it was targeted to collect specimens from 120 mother-child-pairs in each of the 17 participating European countries. These specimens were investigated for six biomarkers (mercury in hair; creatinine, cotinine, cadmium, phthalate metabolites and bisphenol A in urine). The results for mercury in hair are described in a separate paper. Each participating member state was requested to contract laboratories, for capacity building reasons ideally within its borders, carrying out the chemical analyses. To ensure comparability of analytical data a Quality Assurance Unit (QAU) was established which provided the participating laboratories with standard operating procedures (SOP) and with control material. This material was specially prepared from native, non-spiked, pooled urine samples and was tested for homogeneity and stability. Four external quality assessment exercises were carried out. Highly esteemed laboratories from all over the world served as reference laboratories. Web conferences after each external quality assessment exercise functioned as a new and effective tool to improve analytical performance, to build capacity and to educate less experienced laboratories. Of the 38 laboratories participating in the quality assurance exercises 14 laboratories qualified for cadmium, 14 for creatinine, 9 for cotinine, 7 for phthalate metabolites and 5 for bisphenol A in urine. In the last of the four external quality assessment exercises the laboratories that qualified for DEMOCOPHES performed the determinations in urine with relative standard deviations (low/high concentration) of 18.0/2.1% for cotinine, 14.8/5.1% for cadmium, 4.7/3.4% for creatinine. Relative standard deviations for the newly emerging biomarkers were higher, with values between 13.5 and 20.5% for bisphenol A and between 18.9 and 45.3% for the phthalate metabolites. Plausibility control of the HBM results of all participating countries disclosed analytical shortcomings in the determination of Cd when using certain ICP/MS methods. Results were corrected by reanalyzes. The COPHES/DEMOCOPHES project for the first time succeeded in performing a harmonized pan-European HBM project. All data raised have to be regarded as utmost reliable according to the highest international state of the art, since highly renowned laboratories functioned as reference laboratories. The procedure described here, that has shown its success, can be used as a blueprint for future transnational, multicentre HBM projects.
Asunto(s)
Compuestos de Bencidrilo/orina , Cadmio/orina , Cotinina/orina , Creatinina/orina , Exposición a Riesgos Ambientales/análisis , Monitoreo del Ambiente , Fenoles/orina , Ácidos Ftálicos/orina , Adulto , Biomarcadores/orina , Niño , Monitoreo del Ambiente/normas , Contaminantes Ambientales/orina , Europa (Continente) , Femenino , Humanos , Internacionalidad , Laboratorios , Madres , Reproducibilidad de los ResultadosRESUMEN
The 20S proteasome is almost exclusively localized within cells. High levels of extracellular proteasomes are also found circulating in the blood plasma of patients suffering from a variety of inflammatory, autoimmune and neoplastic diseases. However, the origin of these proteasomes remained enigmatic. Since the proteome of microparticles, small membrane enclosed vesicles released from cells, was shown to contain proteasomal subunits, we studied whether intact proteasomes are actively released into the extracellular space. Using human primary T lymphocytes stimulated with CaCl2 and the calcium ionophore A23187 to induce membrane blebbing we demonstrate that microparticles contain proteolytically active 20S proteasomes as well as the proteasome activator PA28 and subunits of the 19S proteasome regulator. Furthermore, our experiments reveal that incubation of in vitro generated T lymphocyte-microparticles with sphingomyelinase results in the hydrolysis of the microparticle membranes and subsequent release of proteasomes from the vesicles. Thus, we here show for the first time that functional proteasomes can be exported from activated immune cells by way of microparticles, the dissolution of which may finally lead to the generation of extracellular proteasomes.
Asunto(s)
Micropartículas Derivadas de Células/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Linfocitos T/metabolismo , Células Cultivadas , Humanos , Subunidades de Proteína/metabolismo , Esfingomielina Fosfodiesterasa/química , Linfocitos T/enzimologíaRESUMEN
The aim of this study was to assess the interaction of a series of well characterised nano-objects with the Gram negative bacterium Salmonella typhimurium, and how such an interaction may relate to the potential mutagenicity of nano-objects. Transmission electron microscopy showed that nano-objects (Au-PMA-ATTO NPs, CeO2 NPs, SWCNTs and MWCNTs), as well as CAFs entered S. typhimurium. Only DEPs did not penetrate/enter the bacteria, however, were the only particle stimulus to induce any significant mutagenicity through the Ames test. Comparison with a sophisticated 3D in vitro cell model showed CAFs, DEPs, SWCNTs and MWCNTs to cause a significant increase in mammalian cell proliferation, whilst both the Au-PMA-ATTO NPs and CeO2 NPs had not significant adverse effects. In conclusion, these results indicate that various of different nano-objects are able to penetrate the double-lipid bilayer of Gram negative bacteria, although the Ames test may not be a good indicator for nano-object mutagenicity.
Asunto(s)
Viabilidad Microbiana/efectos de los fármacos , Pruebas de Mutagenicidad/métodos , Nanoestructuras/toxicidad , Investigación Biomédica , Línea Celular , Proliferación Celular/efectos de los fármacos , Humanos , Salmonella typhimurium/efectos de los fármacosRESUMEN
Like ubiquitin (Ub), the ubiquitin-like protein FAT10 can serve as a signal for proteasome-dependent protein degradation. Here, we investigated the contribution of FAT10 substrate modification to MHC class I antigen presentation. We show that N-terminal modification of the human cytomegalovirus-derived pp65 antigen to FAT10 facilitates direct presentation and dendritic cell-mediated cross-presentation of the HLA-A2 restricted pp65(495-503) epitope. Interestingly, our data indicate that the pp65 presentation initiated by either FAT10 or Ub partially relied on the 19S proteasome subunit Rpn10 (S5a). However, FAT10 distinguished itself from Ub in that it promoted a pp65 response which was not influenced by immunoproteasomes or PA28. Further divergence occurred at the level of Ub-binding proteins with NUB1 supporting the pp65 presentation arising from FAT10, while it exerted no effect on that initiated by Ub. Collectively, our data establish FAT10 modification as a distinct and alternative signal for facilitated MHC class I antigen presentation.
Asunto(s)
Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Citomegalovirus/metabolismo , Células HEK293 , Antígeno HLA-A2/inmunología , Antígeno HLA-A2/metabolismo , Células HeLa , Humanos , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma , Proteolisis , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismoRESUMEN
A triple cell co-culture model was recently established by the authors, consisting of either A549 or 16HBE14o- epithelial cells, human blood monocyte-derived macrophages and dendritic cells, which offers the possibility to study the interaction of xenobiotics with those cells. The 16HBE14o- containing co-culture model mimics the airway epithelial barrier, whereas the A549 co-cultures mimic the alveolar type II-like epithelial barrier. The goal of the present work was to establish a new triple cell co-culture model composed of primary alveolar type I-like cells isolated from human lung biopsies (hAEpC) representing a more realistic alveolar epithelial barrier wall, since type I epithelial cells cover >93% of the alveolar surface. Monocultures of A549 and 16HBE14o- were morphologically and functionally compared with the hAEpC using laser scanning microscopy, as well as transmission electron microscopy, and by determining the epithelial integrity. The triple cell co-cultures were characterized using the same methods. It could be shown that the epithelial integrity of hAEpC (mean ± SD, 1180 ± 188 Ω cm(2)) was higher than in A549 (172 ± 59 Ω cm(2)) but similar to 16HBE14o- cells (1469 ± 156 Ω cm(2)). The triple cell co-culture model with hAEpC (1113 ± 30 Ω cm(2)) showed the highest integrity compared to the ones with A549 (93 ± 14 Ω cm(2)) and 16HBE14o- (558 ± 267 Ω cm(2)). The tight junction protein zonula occludens-1 in hAEpC and 16HBE14o- were more regularly expressed but not in A549. The epithelial alveolar model with hAEpC combined with two immune cells (i.e. macrophages and dendritic cells) will offer a novel and more realistic cell co-culture system to study possible cell interactions of inhaled xenobiotics and their toxic potential on the human alveolar type I epithelial wall.
Asunto(s)
Células Epiteliales/citología , Macrófagos/citología , Modelos Biológicos , Alveolos Pulmonares/citología , Transporte Biológico Activo , Comunicación Celular/efectos de los fármacos , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Técnicas de Cocultivo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Células Dendríticas/ultraestructura , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Humanos , Macrófagos/metabolismo , Macrófagos/ultraestructura , Microscopía Confocal , Microscopía Electrónica de Transmisión , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/ultraestructura , Xenobióticos/farmacocinética , Xenobióticos/toxicidadRESUMEN
During pregnancy, trophoblasts grow to adapt the feto-maternal unit to fetal requirements. Aldosterone and cortisol levels increase, the latter being inactivated by a healthy placenta. By contrast, preeclamptic placental growth is reduced while aldosterone levels are low and placental cortisol tissue levels are high due to improper deactivation. Aldosterone acts as a growth factor in many tissues, whereas cortisol inhibits growth. We hypothesized that in preeclampsia low aldosterone and enhanced cortisol availability might mutually affect placental growth and function. Proliferation of cultured human trophoblasts was time- and dose-dependently increased with aldosterone (P < 0.04 to P < 0.0001) and inhibited by spironolactone and glucocorticoids (P < 0.01). Mineralo- and glucocorticoid receptor expression and activation upon agonist stimulation was verified by visualization of nuclear translocation of the receptors. Functional aldosterone deficiency simulated in pregnant mice by spironolactone treatment (15 µg/g body weight/day) led to a reduced fetal umbilical blood flow (P < 0.05). In rat (P < 0.05; R(2) = 0.2055) and human (X(2) = 3.85; P = 0.0249) pregnancy, placental size was positively related to plasma aldosterone. Autocrine production of these steroid hormones was excluded functionally and via the absence of specific enzymatic transcripts for CYP11B2 and CYP11B1. In conclusion, activation of mineralocorticoid receptors by maternal aldosterone appears to be required for trophoblast growth and a normal feto-placental function. Thus, low aldosterone levels and enhanced cortisol availability may be one explanation for the reduced placental size in preeclampsia and related disorders.
Asunto(s)
Aldosterona/metabolismo , Hidrocortisona/metabolismo , Placenta/fisiología , Placentación , Animales , Línea Celular , Femenino , Humanos , Ratones , Embarazo , RatasRESUMEN
The human airway epithelium serves as structural and functional barrier against inhaled particulate antigen. Previously, we demonstrated in an in vitro epithelial barrier model that monocyte derived dendritic cells (MDDC) and monocyte derived macrophages (MDM) take up particulate antigen by building a trans-epithelial interacting network. Although the epithelial tight junction (TJ) belt was penetrated by processes of MDDC and MDM, the integrity of the epithelium was not affected. These results brought up two main questions: (1) Do MDM and MDDC exchange particles? (2) Are those cells expressing TJ proteins, which are believed to interact with the TJ belt of the epithelium to preserve the epithelial integrity? The expression of TJ and adherens junction (AJ) mRNA and proteins in MDM and MDDC monocultures was determined by RT-PCR, and immunofluorescence, respectively. Particle uptake and exchange was quantified by flow cytometry and laser scanning microscopy in co-cultures of MDM and MDDC exposed to polystyrene particles (1 µm in diameter). MDM and MDDC constantly expressed TJ and AJ mRNA and proteins. Flow cytometry analysis of MDM and MDDC co-cultures showed increased particle uptake in MDDC while MDM lost particles over time. Quantitative analysis revealed significantly higher particle uptake by MDDC in co-cultures of epithelial cells with MDM and MDDC present, compared to co-cultures containing only epithelial cells and MDDC. We conclude from these findings that MDM and MDDC express TJ and AJ proteins which could help to preserve the epithelial integrity during particle uptake and exchange across the lung epithelium.