RESUMEN
Temperature above the physiological optimum is a stress condition frequently faced by bacteria in their natural environments. Here, we were interested in the correlation between levels of RNA and protein under heat stress. Changes in RNA and protein levels were documented in cultures of Rhodobacter sphaeroides using RNA sequencing, quantitative mass spectrometry, western blot analysis, in vivo [35 S] methionine-labelling and plasmid-borne reporter fusions. Changes in the transcriptome were extensive. Strikingly, the proteome remained unchanged except for very few proteins. Examples include a heat shock protein, a DUF1127 protein of unknown function and sigma factor proteins from leaderless transcripts. Insight from this study indicates that R. sphaeroides responds to heat stress by producing a broad range of transcripts while simultaneously preventing translation from nearly all of them, and that this selective production of protein depends on the untranslated region of the transcript. We conclude that measurements of transcript abundance are insufficient to understand gene regulation. Rather, translation can be an important checkpoint for protein expression under certain environmental conditions. Furthermore, during heat shock, regulation at the level of transcription might represent preparation for survival in an unpredictable environment while regulation at translation ensures production of only a few proteins.
Asunto(s)
Rhodobacter sphaeroides , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Respuesta al Choque Térmico/genética , Proteómica , Rhodobacter sphaeroides/genética , Factor sigma/metabolismoRESUMEN
Cardiac metabolism plays a crucial role in producing sufficient energy to sustain cardiac function. However, the role of metabolism in different aspects of cardiomyocyte regeneration remains unclear. Working with the adult zebrafish heart regeneration model, we first find an increase in the levels of mRNAs encoding enzymes regulating glucose and pyruvate metabolism, including pyruvate kinase M1/2 (Pkm) and pyruvate dehydrogenase kinases (Pdks), especially in tissues bordering the damaged area. We further find that impaired glycolysis decreases the number of proliferating cardiomyocytes following injury. These observations are supported by analyses using loss-of-function models for the metabolic regulators Pkma2 and peroxisome proliferator-activated receptor gamma coactivator 1 alpha. Cardiomyocyte-specific loss- and gain-of-function manipulations of pyruvate metabolism using Pdk3 as well as a catalytic subunit of the pyruvate dehydrogenase complex (PDC) reveal its importance in cardiomyocyte dedifferentiation and proliferation after injury. Furthermore, we find that PDK activity can modulate cell cycle progression and protrusive activity in mammalian cardiomyocytes in culture. Our findings reveal new roles for cardiac metabolism and the PDK-PDC axis in cardiomyocyte behavior following cardiac injury.
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Miocitos Cardíacos , Pez Cebra , Animales , Proliferación Celular , Glucólisis , Miocitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Pez Cebra/metabolismoRESUMEN
Photosynthetic bacteria have to deal with the risk of photooxidative stress that occurs in presence of light and oxygen due to the photosensitizing activity of (bacterio-) chlorophylls. Facultative phototrophs of the genus Rhodobacter adapt the formation of photosynthetic complexes to oxygen and light conditions, but cannot completely avoid this stress if environmental conditions suddenly change. R. capsulatus has a stronger pigmentation and faster switches to phototrophic growth than R. sphaeroides. However, its photooxidative stress response has not been investigated. Here, we compare both species by transcriptomics and proteomics, revealing that proteins involved in oxidation-reduction processes, DNA, and protein damage repair play pivotal roles. These functions are likely universal to many phototrophs. Furthermore, the alternative sigma factors RpoE and RpoHII are induced in both species, even though the genetic localization of the rpoE gene, the RpoE protein itself, and probably its regulon, are different. Despite sharing the same habitats, our findings also suggest individual strategies. The crtIB-tspO operon, encoding proteins for biosynthesis of carotenoid precursors and a regulator of photosynthesis, and cbiX, encoding a putative ferrochelatase, are induced in R. capsulatus. This specific response might support adaptation by maintaining high carotenoid-to-bacteriochlorophyll ratios and preventing the accumulation of porphyrin-derived photosensitizers.
RESUMEN
Primary familial brain calcification (PFBC) is an age-dependent and rare neurodegenerative disorder characterized by microvascular calcium phosphate deposits in the deep brain regions. Known genetic causes of PFBC include loss-of-function mutations in genes involved in either of three processes-platelet-derived growth factor (PDGF) signaling, phosphate homeostasis or protein glycosylation-with unclear molecular links. To provide insight into the pathogenesis of PFBC, we analyzed murine models of PFBC for the first two of these processes in Pdgfbret/ret and Slc20a2-/- mice with regard to the structure, molecular composition, development and distribution of perivascular calcified nodules. Analyses by transmission electron microscopy and immunofluorescence revealed that calcified nodules in both of these models have a multilayered ultrastructure and occur in direct contact with reactive astrocytes and microglia. However, whereas nodules in Pdgfbret/ret mice were large, solitary and smooth surfaced, the nodules in Slc20a2-/- mice were multi-lobulated and occurred in clusters. The regional distribution of nodules also differed between the two models. Proteomic analysis and immunofluorescence stainings revealed a common molecular composition of the nodules in the two models, involving proteins implicated in bone homeostasis, but also proteins not previously linked to tissue mineralization. While the brain vasculature of Pdgfbret/ret mice has been reported to display reduced pericyte coverage and abnormal permeability, we found that Slc20a2-/- mice have a normal pericyte coverage and no overtly increased permeability. Thus, lack of pericytes and increase in permeability of the blood-brain barrier are likely not the causal triggers for PFBC pathogenesis. Instead, gene expression and spatial correlations suggest that astrocytes are intimately linked to the calcification process in PFBC.
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Astrocitos/metabolismo , Encefalopatías/metabolismo , Calcinosis/metabolismo , Matriz Extracelular/metabolismo , Microglía/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Animales , Astrocitos/patología , Encefalopatías/genética , Encefalopatías/patología , Calcinosis/genética , Calcinosis/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Matriz Extracelular/patología , Femenino , Masculino , Ratones , Ratones Transgénicos , Microglía/patología , Mutación , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/metabolismoRESUMEN
Exhaustion of nutritional resources stimulates bacterial populations to adapt their growth behaviour. General mechanisms are known to facilitate this adaptation by sensing the environmental change and coordinating gene expression. However, the existence of such mechanisms among the Alphaproteobacteria remains unclear. This study focusses on global changes in transcript levels during growth under carbon-limiting conditions in a model Alphaproteobacterium, Rhodobacter sphaeroides, a metabolically diverse organism capable of multiple modes of growth including aerobic and anaerobic respiration, anaerobic anoxygenic photosynthesis and fermentation. We identified genes that showed changed transcript levels independently of oxygen levels during the adaptation to stationary phase. We selected a subset of these genes and subjected them to mutational analysis, including genes predicted to be involved in manganese uptake, polyhydroxybutyrate production and quorum sensing and an alternative sigma factor. Although these genes have not been previously associated with the adaptation to stationary phase, we found that all were important to varying degrees. We conclude that while R. sphaeroides appears to lack a rpoS-like master regulator of stationary phase adaptation, this adaptation is nonetheless enabled through the impact of multiple genes, each responding to environmental conditions and contributing to the adaptation to stationary phase.
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Adaptación Fisiológica , Rhodobacter sphaeroides/fisiología , Proteínas Bacterianas/genética , Ciclo Celular , Regulación Bacteriana de la Expresión Génica , Rhodobacter sphaeroides/genética , Factor sigma/genéticaRESUMEN
Mechanical forces regulate cell behavior and tissue morphogenesis. During cardiac development, mechanical stimuli from the heartbeat are required for cardiomyocyte maturation, but the underlying molecular mechanisms remain unclear. Here, we first show that the forces of the contracting heart regulate the localization and activation of the cytoskeletal protein vinculin (VCL), which we find to be essential for myofilament maturation. To further analyze the role of VCL in this process, we examined its interactome in contracting versus non-contracting cardiomyocytes and, in addition to several known interactors, including actin regulators, identified the slingshot protein phosphatase SSH1. We show how VCL recruits SSH1 and its effector, the actin depolymerizing factor cofilin (CFL), to regulate F-actin rearrangement and promote cardiomyocyte myofilament maturation. Overall, our results reveal that mechanical forces generated by cardiac contractility regulate cardiomyocyte maturation through the VCL-SSH1-CFL axis, providing further insight into how mechanical forces are transmitted intracellularly to regulate myofilament maturation.
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Cofilina 1/metabolismo , Corazón/embriología , Miocitos Cardíacos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Vinculina/metabolismo , Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Aminobenzoatos/farmacología , Animales , Regulación del Desarrollo de la Expresión Génica , Proteínas de Microfilamentos/metabolismo , Miocardio/metabolismo , Miofibrillas/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Pez CebraRESUMEN
BACKGROUND: In natural environments, bacteria must frequently cope with extremely scarce nutrients. Most studies focus on bacterial growth in nutrient replete conditions, while less is known about the stationary phase. Here, we are interested in global gene expression throughout all growth phases, including the adjustment to deep stationary phase. RESULTS: We monitored both the transcriptome and the proteome in cultures of the alphaproteobacterium Rhodobacter sphaeroides, beginning with the transition to stationary phase and at different points of the stationary phase and finally during exit from stationary phase (outgrowth) following dilution with fresh medium. Correlation between the transcriptomic and proteomic changes was very low throughout the growth phases. Surprisingly, even in deep stationary phase, the abundance of many proteins continued to adjust, while the transcriptome analysis revealed fewer adjustments. This pattern was reversed during the first 90 min of outgrowth, although this depended upon the duration of the stationary phase. We provide a detailed analysis of proteomic changes based on the clustering of orthologous groups (COGs), and compare these with the transcriptome. CONCLUSIONS: The low correlation between transcriptome and proteome supports the view that post-transcriptional processes play a major role in the adaptation to growth conditions. Our data revealed that many proteins with functions in transcription, energy production and conversion and the metabolism and transport of amino acids, carbohydrates, lipids, and secondary metabolites continually increased in deep stationary phase. Based on these findings, we conclude that the bacterium responds to sudden changes in environmental conditions by a radical and rapid reprogramming of the transcriptome in the first 90 min, while the proteome changes were modest. In response to gradually deteriorating conditions, however, the transcriptome remains mostly at a steady state while the bacterium continues to adjust its proteome. Even long after the population has entered stationary phase, cells are still actively adjusting their proteomes.
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Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Variación Genética , Proteoma/análisis , Rhodobacter sphaeroides/crecimiento & desarrollo , Transcriptoma , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismoRESUMEN
BACKGROUND: Chronic heart failure (CHF) leads to diaphragm myopathy that significantly impairs quality of life and worsens prognosis. In this study, we aimed to assess the efficacy of a recently discovered small-molecule inhibitor of MuRF1 in treating CHF-induced diaphragm myopathy and loss of contractile function. METHODS: Myocardial infarction was induced in mice by ligation of the left anterior descending coronary artery. Sham-operated animals (sham) served as controls. One week post-left anterior descending coronary artery ligation animals were randomized into two groups-one group was fed control rodent chow, whereas the other group was fed a diet containing 0.1% of the compound ID#704946-a recently described MuRF1-interfering small molecule. Echocardiography confirmed development of CHF after 10 weeks. Functional and molecular analysis of the diaphragm was subsequently performed. RESULTS: Chronic heart failure induced diaphragm fibre atrophy and contractile dysfunction by ~20%, as well as decreased activity of enzymes involved in mitochondrial energy production (P < 0.05). Treatment with compound ID#704946 in CHF mice had beneficial effects on the diaphragm: contractile function was protected, while mitochondrial enzyme activity and up-regulation of the MuRF1 and MuRF2 was attenuated after infarct. CONCLUSIONS: Our murine CHF model presented with diaphragm fibre atrophy, impaired contractile function, and reduced mitochondrial enzyme activities. Compound ID#704946 rescued from this partially, possibly by targeting MuRF1/MuRF2. However, at this stage of our study, we refrain to claim specific mechanism(s) and targets of compound ID#704946, because the nature of changes after 12 weeks of feeding is likely to be complex and is not necessarily caused by direct mechanistic effects.
Asunto(s)
Diafragma/metabolismo , Diafragma/fisiopatología , Insuficiencia Cardíaca/complicaciones , Proteínas Musculares/antagonistas & inhibidores , Proteínas de Motivos Tripartitos/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Animales , Línea Celular , Enfermedad Crónica , Diafragma/efectos de los fármacos , Ecocardiografía , Femenino , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Humanos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Contracción Muscular/efectos de los fármacos , Proteómica/métodosRESUMEN
Down syndrome (DS) or trisomy 21 (T21) is a leading genetic cause of intellectual disability. To gain insights into dynamics of molecular perturbations during neurogenesis in DS, we established a model using induced pluripotent stem cells (iPSC) with transcriptome profiles comparable to that of normal fetal brain development. When applied on iPSCs with T21, transcriptome and proteome signatures at two stages of differentiation revealed strong temporal dynamics of dysregulated genes, proteins and pathways belonging to 11 major functional clusters. DNA replication, synaptic maturation and neuroactive clusters were disturbed at the early differentiation time point accompanied by a skewed transition from the neural progenitor cell stage and reduced cellular growth. With differentiation, growth factor and extracellular matrix, oxidative phosphorylation and glycolysis emerged as major perturbed clusters. Furthermore, we identified a marked dysregulation of a set of genes encoded by chromosome 21 including an early upregulation of the hub gene APP, supporting its role for disturbed neurogenesis, and the transcription factors OLIG1, OLIG2 and RUNX1, consistent with deficient myelination and neuronal differentiation. Taken together, our findings highlight novel sequential and differentiation-dependent dynamics of disturbed functions, pathways and elements in T21 neurogenesis, providing further insights into developmental abnormalities of the DS brain.
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Síndrome de Down/genética , Síndrome de Down/patología , Células Madre Pluripotentes Inducidas/patología , Neuronas/metabolismo , Neuronas/patología , Proteoma/metabolismo , Transcriptoma/genética , Diferenciación Celular/genética , Proliferación Celular/genética , Femenino , Humanos , Masculino , Mitocondrias/genética , Modelos Biológicos , Neuritas/metabolismo , Neurogénesis/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo , Transcripción GenéticaRESUMEN
Bacterial populations produce phenotypic variants called persisters to survive harmful conditions. Persisters are highly tolerant to antibiotics and repopulate environments after the stress has vanished. In order to resume growth, persisters have to recover from the persistent state, but the processes behind recovery remain mostly elusive. Deciphering these processes is an essential step toward understanding the persister phenomenon in its entirety. High-throughput proteomics by mass spectrometry is a valuable tool to assess persister physiology during any stage of the persister life cycle, and is expected to considerably contribute to our understanding of the recovery process. In the present study, an Escherichia coli strain, that overproduces the membrane-depolarizing toxin TisB, was established as a model for persistence by the use of high-throughput proteomics. Labeling of TisB persisters with stable isotope-containing amino acids (pulsed-SILAC) revealed an active translational response to ampicillin, including several RpoS-dependent proteins. Subsequent investigation of the persister proteome during postantibiotic recovery by label-free quantitative proteomics identified proteins with importance to the recovery process. Among them, AhpF, a component of alkyl hydroperoxide reductase, and the outer membrane porin OmpF were found to affect the persistence time of TisB persisters. Assessing the role of AhpF and OmpF in TisB-independent persisters demonstrated that the importance of a particular protein for the recovery process strongly depends on the physiological condition of a persister cell. Our study provides important insights into persister physiology and the processes behind recovery of depolarized cells.
RESUMEN
BACKGROUND/AIMS: Pseudoexfoliation syndrome (PEX) is characterised by the production and accumulation of extracellular fibrillar material in the anterior segment of the eye. The pathogenesis of PEX is multifactorial with genetic factors and ageing as contributing factors. Previously, an increased concentration of beta-crystalline B2 (CRYBB2) was observed in the aqueous humour (AH) in eyes with PEX in a pooled material. Here, the protein content was examined on individual basis. METHODS: During cataract surgery, AH was sampled from patients with and without PEX, 10 eyes in each group. The proteins were digested and labelled with isotopomeric dimethyl labels, separated with high-pressure liquid chromatography and analysed in an Orbitrap mass analyzer. RESULTS: The concentration of complement factor 3, kininogen-1, antithrombin III and vitamin D-binding protein was increased in all eyes with PEX. Retinol-binding protein 3, glutathione peroxidase, calsyntenin-1 and carboxypeptidase E were decreased in eyes with PEX. Beta-crystalline B1 and CRYBB2 and gamma-crystalline D were up to eightfold upregulated in 4 of 10 in eyes with PEX. CONCLUSION : The results indicate that oxidative stress and inflammation are contributing factors in the formation of PEX. Knowledge about the proteome in PEX is relevant for understanding this condition.
Asunto(s)
Humor Acuoso/metabolismo , Síndrome de Exfoliación/diagnóstico , Proteínas del Ojo/análisis , Proteómica/métodos , Anciano , Anciano de 80 o más Años , Biomarcadores/análisis , Síndrome de Exfoliación/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los ResultadosRESUMEN
The peritoneal fluid of ovarian carcinoma patients promotes cancer cell invasion and metastatic spread with lysophosphatidic acid (LPA) as a potentially crucial mediator. However, the origin of LPA in ascites and the clinical relevance of individual LPA species have not been addressed. Here, we show that the levels of multiple acyl-LPA species are strongly elevated in ascites versus plasma and are associated with short relapse-free survival. Data derived from transcriptome and secretome analyses of primary ascite-derived cells indicate that (a) the major route of LPA synthesis is the consecutive action of a secretory phospholipase A2 (PLA2 ) and autotaxin, (b) that the components of this pathway are coordinately upregulated in ascites, and (c) that CD163+CD206+ tumor-associated macrophages play an essential role as main producers of PLA2 G7 and autotaxin. The latter conclusion is consistent with mass spectrometry-based metabolomic analyses of conditioned medium from ascites cells, which showed that tumor-associated macrophages, but not tumor cells, are able to produce 20:4 acyl-LPA in lipid-free medium. Furthermore, our transcriptomic data revealed that LPA receptor (LPAR) genes are expressed in a clearly cell type-selective manner: While tumor cells express predominantly LPAR1-3, macrophages and T cells also express LPAR5 and LPAR6 at high levels, pointing to cell type-selective LPA signaling pathways. RNA profiling identified cytokines linked to cell motility and migration as the most conspicuous class of LPA-induced genes in macrophages, suggesting that LPA exerts protumorigenic properties at least in part via the tumor secretome.
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Lisofosfolípidos/biosíntesis , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Transducción de Señal , Microambiente Tumoral , Ascitis/metabolismo , Línea Celular Tumoral , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Metaboloma , Neoplasias Quísticas, Mucinosas y Serosas/patología , Neoplasias Ováricas/genética , Receptores del Ácido Lisofosfatídico/metabolismo , Resultado del Tratamiento , Microambiente Tumoral/genética , Regulación hacia Arriba/genéticaRESUMEN
Ovarian cancer is characterized by early transcoelomic metastatic spread via the peritoneal fluid, where tumor cell spheroids (TU), tumor-associated T cells (TAT), and macrophages (TAM) create a unique microenvironment promoting cancer progression, chemoresistance, and immunosuppression. However, the underlying signaling mechanisms remain largely obscure. To chart these signaling networks, we performed comprehensive proteomic and transcriptomic analyses of TU, TAT, and TAM from ascites of ovarian cancer patients. We identify multiple intercellular signaling pathways driven by protein or lipid mediators that are associated with clinical outcome. Beyond cytokines, chemokines and growth factors, these include proteins of the extracellular matrix, immune checkpoint regulators, complement factors, and a prominent network of axon guidance molecules of the ephrin, semaphorin, and slit families. Intriguingly, both TU and TAM from patients with a predicted short survival selectively produce mediators supporting prometastatic events, including matrix remodeling, stemness, invasion, angiogenesis, and immunosuppression, whereas TAM associated with a longer survival express cytokines linked to effector T-cell chemoattraction and activation. In summary, our study uncovers previously unrecognized signaling networks in the ovarian cancer microenvironment that are of potential clinical relevance.
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Proteínas de Neoplasias/metabolismo , Neoplasias Ováricas/metabolismo , Microambiente Tumoral , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Proteómica , Transducción de SeñalRESUMEN
The proteome and phosphoproteome of non-structural proteins of Adenovirus type 2 (Ad2) were time resolved using a developed mass spectrometry approach. These proteins are expressed by the viral genome and important for the infection process, but not part of the virus particle. We unambiguously confirm the existence of 95% of the viral proteins predicted to be encoded by the viral genome. Most non-structural proteins peaked in expression at late time post infection. We identified 27 non-redundant sites of phosphorylation on seven different non-structural proteins. The most heavily phosphorylated protein was the DNA binding protein (DBP) with 15 different sites. The phosphorylation occupancy rate could be calculated and monitored with time post infection for 15 phosphorylated sites on various proteins. In the DBP, phosphorylations with time-dependent relation were observed. The findings show the complexity of the Ad2 non-structural proteins and opens up a discussion for potential new drug targets.
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Adenovirus Humanos/crecimiento & desarrollo , Regulación Viral de la Expresión Génica , Fosfoproteínas/análisis , Proteoma/análisis , Proteínas no Estructurales Virales/análisis , Línea Celular , Fibroblastos/virología , Humanos , Espectrometría de Masas , Factores de TiempoRESUMEN
A hitherto predicted but undetected protein, C-168, in adenovirus type 2 (Ad2) has been identified using mass spectrometry (MS) based proteomics. The gene of this 17.7kDa protein is located on the forward strand in the major late transcription unit between base pairs 9294 and 9797. A tryptic peptide, derived from the C-terminal part of the protein, was identified with high amino acid sequence coverage. A candidate splice site for the corresponding mRNA is also presented. The protein sequence is unusual with repeats of serine, glycine and arginine. A bioinformatics prediction of protein function and localization is presented.
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Adenovirus Humanos/química , Proteínas Virales/análisis , Adenovirus Humanos/genética , Espectrometría de Masas , Peso Molecular , Proteínas Virales/genéticaRESUMEN
The small ubiquitin-like modifier (SUMO) is as a regulator of many cellular functions by reversible conjugation to a broad number of substrates. Under endogenous or exogenous perturbations, the SUMO network becomes a fine sensor of stress conditions by alterations in the expression level of SUMO enzymes and consequently changing the status of SUMOylated proteins. The diaphragm is the major inspiratory muscle, which is continuously active under physiological conditions, but its structure and function is severely affected when passively displaced for long extents during mechanical ventilation (MV). An iatrogenic condition called Ventilator-Induced Diaphragm Dysfunction (VIDD) is a major cause of failure to wean patients from ventilator support but the molecular mechanisms underlying this dysfunction are not fully understood. Using a unique experimental Intensive Care Unit (ICU) rat model allowing long-term MV, diaphragm muscles were collected in rats control and exposed to controlled MV (CMV) for durations varying between 1 and 10 days. Endogenous SUMOylated diaphragm proteins were identified by mass spectrometry and validated with in vitro SUMOylation systems. Contractile, calcium regulator and mitochondrial proteins were of specific interest due to their putative involvement in VIDD. Differences were observed in the abundance of SUMOylated proteins between glycolytic and oxidative muscle fibers in control animals and high levels of SUMOylated proteins were present in all fibers during CMV. Finally, previously reported VIDD biomarkers and therapeutic targets were also identified in our datasets which may play an important role in response to muscle weakness seen in ICU patients. Data are available via ProteomeXchange with identifier PXD006085. Username: reviewer26663@ebi.ac.uk, Password: rwcP5W0o.
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Diafragma/metabolismo , Respiración Artificial , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Animales , Sedación Profunda , Femenino , Bloqueo Neuromuscular , Proteómica , Ratas Sprague-DawleyRESUMEN
A deeper understanding of how viruses reprogram their hosts for production of progeny is needed to combat infections. Most knowledge on the regulation of cellular gene expression during adenovirus infection is derived from mRNA studies. Here, we investigated the changes in protein expression during the late phase of adenovirus type 2 (Ad2) infection of the IMR-90 cell line by stable isotope labeling in cell culture with subsequent liquid chromatography-high resolution tandem mass spectrometric analysis. Two biological replicates of samples collected at 24 and 36 h post-infection (hpi) were investigated using swapped labeling. In total, 2648 and 2394 proteins were quantified at 24 and 36 hpi, respectively. Among them, 659 and 645 were deregulated >1.6-fold at the two time points. The protein expression was compared with RNA expression using cDNA sequencing data. The correlation was surprisingly low (r = 0.3), and several examples of posttranscriptional regulation were observed; e.g., proteins related to carbohydrate metabolism were up-regulated at the protein level but unchanged at the RNA level, whereas histone proteins were down-regulated at the protein level but up-regulated at the RNA level. The deregulation of cellular gene expression by adenovirus is mediated at multiple levels and more complex than hitherto believed.
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Adenoviridae/fisiología , Interacciones Huésped-Patógeno , Miofibroblastos/metabolismo , Proteoma/genética , Procesamiento Postranscripcional del ARN , ARN/biosíntesis , Metabolismo de los Hidratos de Carbono/genética , Línea Celular , ADN Complementario/análisis , ADN Complementario/biosíntesis , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ontología de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Histonas/genética , Histonas/metabolismo , Humanos , Marcaje Isotópico , Espectrometría de Masas , Anotación de Secuencia Molecular , Miofibroblastos/virología , Proteoma/metabolismoRESUMEN
Phosphorylation of proteins is important for controlling cellular signaling and cell cycle regulatory events. The process is reversible and phosphoproteins normally constitute a minor part of the global proteome in a cell. Thus, sample preparation techniques tailored for phosphoproteome studies are continuously invented and evaluated. This paper aims at evaluating the performances of the most popular techniques for phospho-enrichments in sub-proteome analysis, such as viral proteomes expressed in human cells during infection. A two-species sample of Adenovirus type 2 infected human cells was used, and in-solution digestion, strong cation exchange (SCX), and electrostatic repulsion hydrophilic interaction chromatography (ERLIC) fractionation, and subsequent enrichment by TiO2, were compared with SDS-PAGE fractionation and in-gel digestion. Evaluation was focused on phosphopeptide detection in the sub-proteome. The results showed that the SCX+TiO2 or ERLIC+TiO2 combinations had the highest enrichment efficiencies, but SDS-PAGE fractionation and in-gel digestion resulted in the highest number of identified proteins and phosphopeptides. Furthermore, the study demonstrates the usefulness of applying as many orthogonal techniques as possible in deep phosphoproteome analysis, since the overlap between approaches was low.
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Fosfopéptidos/química , Proteoma , Línea Celular , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Humanos , Espectrometría de Masas en TándemRESUMEN
There is increasing evidence that dosage compensation is not a ubiquitous feature following sex chromosome evolution, especially not in organisms where females are the heterogametic sex, like in birds. Even when it occurs, compensation can be incomplete and limited to dosage-sensitive genes. However, previous work has mainly studied transcriptional regulation of sex-linked genes, which may not reflect expression at the protein level. Here, we used liquid chromatography-tandem mass spectrometry to detect and quantify expressed levels of more than 2,400 proteins in ten different tissues of male and female chicken embryos. For comparison, transcriptome sequencing was performed in the same individuals, five of each sex. The proteomic analysis revealed that dosage compensation was incomplete, with a mean male-to-female (M:F) expression ratio of Z-linked genes of 1.32 across tissues, similar to that at the RNA level (1.29). The mean Z chromosome-to-autosome expression ratio was close to 1 in males and lower than 1 in females, consistent with partly reduced Z chromosome expression in females. Although our results exclude a general mechanism for chromosome-wide dosage compensation at translation, 30% of all proteins encoded from Z-linked genes showed a significant change in the M:F ratio compared with the corresponding ratio at the RNA level. This resulted in a pattern where some genes showed balanced expression between sexes and some close to 2-fold higher expression in males. This suggests that proteomic analyses will be necessary to reveal a more complete picture of gene regulation and sex chromosome evolution.
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Pollos/genética , Compensación de Dosificación (Genética) , Regulación de la Expresión Génica , Espectrometría de Masas/métodos , Biosíntesis de Proteínas/genética , Animales , Cromosomas/genética , Femenino , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS) are characterized by neuronal impairment that leads to disease-specific changes in the neuronal proteins. The early diagnosis of these disorders is difficult, thus, the need for identifying, developing and using valid clinically applicable biomarkers that meet the criteria of precision, specificity and repeatability is very vital. The application of rapidly emerging technology such as mass spectrometry (MS) in proteomics has opened new avenues to accelerate biomarker discovery, both for diagnostic as well as for prognostic purposes. This review summarizes the most recent advances in the mass spectrometry-based neuroproteomics and analyses the current and future directions in the biomarker discovery for the neurodegenerative diseases. This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology.