RESUMO
RNA-binding proteins (RBPs) and long non-coding RNAs (lncRNAs) are key regulators of gene expression, but their joint functions in coordinating cell fate decisions are poorly understood. Here we show that the expression and activity of the RBP TDP-43 and the long isoform of the lncRNA Neat1, the scaffold of the nuclear compartment "paraspeckles," are reciprocal in pluripotent and differentiated cells because of their cross-regulation. In pluripotent cells, TDP-43 represses the formation of paraspeckles by enhancing the polyadenylated short isoform of Neat1. TDP-43 also promotes pluripotency by regulating alternative polyadenylation of transcripts encoding pluripotency factors, including Sox2, which partially protects its 3' UTR from miR-21-mediated degradation. Conversely, paraspeckles sequester TDP-43 and other RBPs from mRNAs and promote exit from pluripotency and embryonic patterning in the mouse. We demonstrate that cross-regulation between TDP-43 and Neat1 is essential for their efficient regulation of a broad network of genes and, therefore, of pluripotency and differentiation.
Assuntos
Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias Murinas/metabolismo , RNA Longo não Codificante/genética , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , MicroRNAs/genética , Células-Tronco Pluripotentes/metabolismo , Poliadenilação/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Ribosomes translate mRNA into proteins and are essential for every living organism. In eukaryotes, both ribosomal subunits are rapidly assembled in a strict hierarchical order, starting in the nucleolus with the transcription of a common precursor ribosomal RNA (pre-rRNA). This pre-rRNA encodes three of the four mature rRNAs, which are formed by several, consecutive endonucleolytic and exonucleolytic processing steps. Historically, northern blots are used to analyze the variety of different pre-rRNA species, only allowing rough length estimations. Although this limitation can be overcome with primer extension, both approaches often use radioactivity and are time-consuming and costly. Here, we present "Riboprobing," a linker ligation-based workflow followed by reverse transcription and PCR for easy and fast detection and characterization of pre-rRNA species and their 5' as well as 3' ends. Using standard molecular biology laboratory equipment, "Riboprobing" allows reliable discrimination of pre-rRNA species not resolved by northern blot (e.g., 27SA2, 27SA3, and 27SB pre-rRNA). The method can successfully be used for the analysis of total cell extracts as well as purified pre-ribosomes for a straightforward evaluation of the impact of mutant gene versions or inhibitors. In the course of method development, we identified and characterized a hitherto undescribed aberrant pre-rRNA arising from LiCl inhibition. This pre-rRNA fragment spans from processing site A1 to E, forming a small RNP that lacks most early joining assembly factors. This finding expands our knowledge of how the cell deals with severe pre-rRNA processing defects and demonstrates the strict requirement for the 5'ETS (external transcribed spacer) for the assembly process.
Assuntos
Precursores de RNA , RNA Ribossômico , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Fluxo de Trabalho , Processamento Pós-Transcricional do RNARESUMO
The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors1 that delaminate and settle in the subventricular zone in enlarged brain regions2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination.
Assuntos
Centrossomo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ventrículos Laterais/citologia , Ventrículos Laterais/embriologia , Microtúbulos/metabolismo , Neurogênese , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Movimento Celular , Células Cultivadas , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Humanos , Junções Intercelulares/metabolismo , Interfase , Ventrículos Laterais/anatomia & histologia , Glândulas Mamárias Animais/citologia , Camundongos , Tamanho do Órgão , Organoides/citologiaRESUMO
The RNA-binding protein PURA has been implicated in the rare, monogenetic, neurodevelopmental disorder PURA Syndrome. PURA binds both DNA and RNA and has been associated with various cellular functions. Only little is known about its main cellular roles and the molecular pathways affected upon PURA depletion. Here, we show that PURA is predominantly located in the cytoplasm, where it binds to thousands of mRNAs. Many of these transcripts change abundance in response to PURA depletion. The encoded proteins suggest a role for PURA in immune responses, mitochondrial function, autophagy and processing (P)-body activity. Intriguingly, reduced PURA levels decrease the expression of the integral P-body components LSM14A and DDX6 and strongly affect P-body formation in human cells. Furthermore, PURA knockdown results in stabilization of P-body-enriched transcripts, whereas other mRNAs are not affected. Hence, reduced PURA levels, as reported in patients with PURA Syndrome, influence the formation and composition of this phase-separated RNA processing machinery. Our study proposes PURA Syndrome as a new model to study the tight connection between P-body-associated RNA regulation and neurodevelopmental disorders.
Assuntos
Proteínas de Ligação a RNA , Fatores de Transcrição , Humanos , Proteínas de Ligação a DNA/genética , Epilepsia , Corpos de Processamento , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The foundation for integrating mass spectrometry (MS)-based proteomics into systems medicine is the development of standardized start-to-finish and fit-for-purpose workflows for clinical specimens. An essential step in this pursuit is to highlight the common ground in a diverse landscape of different sample preparation techniques and liquid chromatography-mass spectrometry (LC-MS) setups. With the aim to benchmark and improve the current best practices among the proteomics MS laboratories of the CLINSPECT-M consortium, we performed two consecutive round-robin studies with full freedom to operate in terms of sample preparation and MS measurements. The six study partners were provided with two clinically relevant sample matrices: plasma and cerebrospinal fluid (CSF). In the first round, each laboratory applied their current best practice protocol for the respective matrix. Based on the achieved results and following a transparent exchange of all lab-specific protocols within the consortium, each laboratory could advance their methods before measuring the same samples in the second acquisition round. Both time points are compared with respect to identifications (IDs), data completeness, and precision, as well as reproducibility. As a result, the individual performances of participating study centers were improved in the second measurement, emphasizing the effect and importance of the expert-driven exchange of best practices for direct practical improvements.
Assuntos
Plasma , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Fluxo de Trabalho , Reprodutibilidade dos Testes , Plasma/químicaRESUMO
BACKGROUND: Growing up on traditional European or US Amish dairy farms in close contact with cows and hay protects children against asthma, and airway administration of extracts from dust collected from cowsheds of those farms prevents allergic asthma in mice. OBJECTIVES: This study sought to begin identifying farm-derived asthma-protective agents. METHODS: Our work unfolded along 2 unbiased and independent but complementary discovery paths. Dust extracts (DEs) from protective and nonprotective farms (European and Amish cowsheds vs European sheep sheds) were analyzed by comparative nuclear magnetic resonance profiling and differential proteomics. Bioactivity-guided size fractionation focused on protective Amish cowshed DEs. Multiple in vitro and in vivo functional assays were used in both paths. Some of the proteins thus identified were characterized by in-solution and in-gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis enzymatic digestion/peptide mapping followed by liquid chromatography/mass spectrometry. The cargo carried by these proteins was analyzed by untargeted liquid chromatography-high-resolution mass spectrometry. RESULTS: Twelve carrier proteins of animal and plant origin, including the bovine lipocalins Bos d 2 and odorant binding protein, were enriched in DEs from protective European cowsheds. A potent asthma-protective fraction of Amish cowshed DEs (≈0.5% of the total carbon content of unfractionated extracts) contained 7 animal and plant proteins, including Bos d 2 and odorant binding protein loaded with fatty acid metabolites from plants, bacteria, and fungi. CONCLUSIONS: Animals and plants from traditional farms produce proteins that transport hydrophobic microbial and plant metabolites. When delivered to mucosal surfaces, these agents might regulate airway responses.
Assuntos
Asma , Poeira , Feminino , Animais , Bovinos , Camundongos , Ovinos , Fazendas , Poeira/análise , Asma/prevenção & controle , Alérgenos , Sistema RespiratórioRESUMO
Collagen VI is a key component of muscle basement membranes, and genetic variants can cause monogenic muscular dystrophies. Conversely, human genetic studies recently implicated collagen VI in central nervous system function, with variants causing the movement disorder dystonia. To elucidate the neurophysiological role of collagen VI, we generated mice with a truncation of the dystonia-related collagen α3 VI (COL6A3) C-terminal domain (CTD). These Col6a3CTT mice showed a recessive dystonia-like phenotype in both sexes. We found that COL6A3 interacts with the cannabinoid receptor 1 (CB1R) complex in a CTD-dependent manner. Col6a3CTT mice of both sexes have impaired homeostasis of excitatory input to the basal pontine nuclei (BPN), a motor control hub with dense COL6A3 expression, consistent with deficient endocannabinoid (eCB) signaling. Aberrant synaptic input in the BPN was normalized by a CB1R agonist, and motor performance in Col6a3CTT mice of both sexes was improved by CB1R agonist treatment. Our findings identify a readily therapeutically addressable synaptic mechanism for motor control.SIGNIFICANCE STATEMENT Dystonia is a movement disorder characterized by involuntary movements. We previously identified genetic variants affecting a specific domain of the COL6A3 protein as a cause of dystonia. Here, we created mice lacking the affected domain and observed an analogous movement disorder. Using a protein interaction screen, we found that the affected COL6A3 domain mediates an interaction with the cannabinoid receptor 1 (CB1R). Concordantly, our COL6A3-deficient mice showed a deficit in synaptic plasticity linked to a deficit in cannabinoid signaling. Pharmacological cannabinoid augmentation rescued the motor impairment of the mice. Thus, cannabinoid augmentation could be a promising avenue for treating dystonia, and we have identified a possible molecular mechanism mediating this.
Assuntos
Canabinoides , Colágeno Tipo VI , Distonia , Distúrbios Distônicos , Neurônios Motores , Plasticidade Neuronal , Animais , Canabinoides/metabolismo , Canabinoides/farmacologia , Colágeno Tipo VI/genética , Colágeno Tipo VI/metabolismo , Distonia/genética , Distonia/metabolismo , Distúrbios Distônicos/genética , Distúrbios Distônicos/metabolismo , Feminino , Masculino , Camundongos , Neurônios Motores/efeitos dos fármacos , Mutação , Plasticidade Neuronal/efeitos dos fármacos , Receptores de Canabinoides/genética , Receptores de Canabinoides/metabolismoRESUMO
The human macula is a highly specialized retinal region with pit-like morphology and rich in cones. How Müller cells, the principal glial cell type in the retina, are adapted to this environment is still poorly understood. We compared proteomic data from cone- and rod-rich retinae from human and mice and identified different expression profiles of cone- and rod-associated Müller cells that converged on pathways representing extracellular matrix and cell adhesion. In particular, epiplakin (EPPK1), which is thought to play a role in intermediate filament organization, was highly expressed in macular Müller cells. Furthermore, EPPK1 knockout in a human Müller cell-derived cell line led to a decrease in traction forces as well as to changes in cell size, shape, and filopodia characteristics. We here identified EPPK1 as a central molecular player in the region-specific architecture of the human retina, which likely enables specific functions under the immense mechanical loads in vivo.
Assuntos
Células Ependimogliais , Proteoma , Humanos , Camundongos , Animais , Proteoma/metabolismo , Proteômica , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones , Neuroglia/metabolismoRESUMO
Hybrid-poplar tree plantations provide a source for biofuel and biomass, but they also increase forest isoprene emissions. The consequences of increased isoprene emissions include higher rates of tropospheric ozone production, increases in the lifetime of methane, and increases in atmospheric aerosol production, all of which affect the global energy budget and/or lead to the degradation of air quality. Using RNA interference (RNAi) to suppress isoprene emission, we show that this trait, which is thought to be required for the tolerance of abiotic stress, is not required for high rates of photosynthesis and woody biomass production in the agroforest plantation environment, even in areas with high levels of climatic stress. Biomass production over 4 y in plantations in Arizona and Oregon was similar among genetic lines that emitted or did not emit significant amounts of isoprene. Lines that had substantially reduced isoprene emission rates also showed decreases in flavonol pigments, which reduce oxidative damage during extremes of abiotic stress, a pattern that would be expected to amplify metabolic dysfunction in the absence of isoprene production in stress-prone climate regimes. However, compensatory increases in the expression of other proteomic components, especially those associated with the production of protective compounds, such as carotenoids and terpenoids, and the fact that most biomass is produced prior to the hottest and driest part of the growing season explain the observed pattern of high biomass production with low isoprene emission. Our results show that it is possible to reduce the deleterious influences of isoprene on the atmosphere, while sustaining woody biomass production in temperate agroforest plantations.
Assuntos
Atmosfera , Hemiterpenos/biossíntese , Hibridização Genética , Populus/crescimento & desenvolvimento , Populus/metabolismo , Poluição do Ar , Arizona , Biocombustíveis , Biomassa , Butadienos , Dióxido de Carbono/metabolismo , Carotenoides/metabolismo , Clima , Oregon , Fotossíntese , Folhas de Planta/metabolismo , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Populus/genética , Proteoma , Interferência de RNA , Estações do Ano , Estresse Fisiológico , Terpenos/metabolismo , Termotolerância/fisiologia , MadeiraRESUMO
BACKGROUND: Immune cells contain a specialised type of proteasome, i.e. the immunoproteasome, which is required for intracellular protein degradation. Immunoproteasomes are key regulators of immune cell differentiation, inflammatory activation and autoimmunity. Immunoproteasome function in peripheral immune cells might be altered by smoking and in chronic obstructive pulmonary disease (COPD), thereby affecting immune cell responses. METHODS: We analysed the expression and activity of proteasome complexes in peripheral blood mononuclear cells (PBMCs) isolated from healthy male young smokers as well as from patients with severe COPD and compared them with matching controls. RESULTS: Proteasome expression was upregulated in COPD patients as assessed by quantitative reverse transcriptase-PCR and mass spectrometry-based proteomic analysis. Proteasome activity was quantified using activity-based probes and native gel analysis. We observed distinct activation of immunoproteasomes in the peripheral blood cells of young male smokers and severely ill COPD patients. Native gel analysis and linear regression modelling confirmed robust activation and elevated assembly of 20S proteasomes, which correlated significantly with reduced lung function parameters in COPD patients. The immunoproteasome was distinctly activated in COPD patients upon inflammatory cytokine stimulation of PBMCs in vitro. Inhibition of the immunoproteasome reduced pro-inflammatory cytokine expression in COPD-derived blood immune cells. CONCLUSIONS: Given the crucial role of chronic inflammatory signalling and the emerging involvement of autoimmune responses in COPD, therapeutic targeting of the immunoproteasome might represent a novel therapeutic concept for COPD.
Assuntos
Complexo de Endopeptidases do Proteassoma , Doença Pulmonar Obstrutiva Crônica , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteômica , FumantesRESUMO
Slow microbial degradation of organic trace chemicals ("micropollutants") has been attributed to either downregulation of enzymatic turnover or rate-limiting substrate supply at low concentrations. In previous biodegradation studies, a drastic decrease in isotope fractionation of atrazine revealed a transition from rate-limiting enzyme turnover to membrane permeation as a bottleneck when concentrations fell below the Monod constant of microbial growth. With degradation of the pollutant 4-chlorophenol (4-CP) by Arthrobacter chlorophenolicus A6, this study targeted a bacterium which adapts its enzyme activity to concentrations. Unlike with atrazine degradation, isotope fractionation of 4-CP increased at lower concentrations, from ε(C) = -1.0 ± 0.5 in chemostats (D = 0.090 h-1, 88 mg L-1) and ε(C) = -2.1 ± 0.5 in batch (c0 = 220 mg L-1) to ε(C) = -4.1 ± 0.2 in chemostats at 90 µg L-1. Surprisingly, fatty acid composition indicated increased cell wall permeability at high concentrations, while proteomics revealed that catabolic enzymes (CphCI and CphCII) were differentially expressed at D = 0.090 h-1. These observations support regulation on the enzyme activity levelâthrough either a metabolic shift between catabolic pathways or decreased enzymatic turnover at low concentrationsâand, hence, reveal an alternative end-member scenario for bacterial adaptation at low concentrations. Including more degrader strains into this multidisciplinary analytical approach offers the perspective to build a knowledge base on bottlenecks of bioremediation at low concentrations that considers bacterial adaptation.
Assuntos
Arthrobacter , Atrazina , Biodegradação Ambiental , Isótopos de Carbono/metabolismo , Fracionamento Químico , Isótopos , Micrococcaceae , FenolRESUMO
Climate change is increasing the frequency and intensity of warming and drought periods around the globe, currently representing a threat to many plant species. Understanding the resistance and resilience of plants to climate change is, therefore, urgently needed. As date palm (Phoenix dactylifera) evolved adaptation mechanisms to a xeric environment and can tolerate large diurnal and seasonal temperature fluctuations, we studied the protein expression changes in leaves, volatile organic compound emissions, and photosynthesis in response to variable growth temperatures and soil water deprivation. Plants were grown under controlled environmental conditions of simulated Saudi Arabian summer and winter climates challenged with drought stress. We show that date palm is able to counteract the harsh conditions of the Arabian Peninsula by adjusting the abundances of proteins related to the photosynthetic machinery, abiotic stress and secondary metabolism. Under summer climate and water deprivation, these adjustments included efficient protein expression response mediated by heat shock proteins and the antioxidant system to counteract reactive oxygen species formation. Proteins related to secondary metabolism were downregulated, except for the P. dactylifera isoprene synthase (PdIspS), which was strongly upregulated in response to summer climate and drought. This study reports, for the first time, the identification and functional characterization of the gene encoding for PdIspS, allowing future analysis of isoprene functions in date palm under extreme environments. Overall, the current study shows that reprogramming of the leaf protein profiles confers the date palm heat- and drought tolerance. We conclude that the protein plasticity of date palm is an important mechanism of molecular adaptation to environmental fluctuations.
Assuntos
Phoeniceae , Secas , Fotossíntese , Folhas de Planta , Arábia Saudita , Estresse FisiológicoRESUMO
Isoprene is a C5 volatile organic compound, which can protect aboveground plant tissue from abiotic stress such as short-term high temperatures and accumulation of reactive oxygen species (ROS). Here, we uncover new roles for isoprene in the plant belowground tissues. By analysing Populus x canescens isoprene synthase (PcISPS) promoter reporter plants, we discovered PcISPS promoter activity in certain regions of the roots including the vascular tissue, the differentiation zone and the root cap. Treatment of roots with auxin or salt increased PcISPS promoter activity at these sites, especially in the developing lateral roots (LR). Transgenic, isoprene non-emitting poplar roots revealed an accumulation of O2- in the same root regions where PcISPS promoter activity was localized. Absence of isoprene emission, moreover, increased the formation of LRs. Inhibition of NAD(P)H oxidase activity suppressed LR development, suggesting the involvement of ROS in this process. The analysis of the fine root proteome revealed a constitutive shift in the amount of several redox balance, signalling and development related proteins, such as superoxide dismutase, various peroxidases and linoleate 9S-lipoxygenase, in isoprene non-emitting poplar roots. Together our results indicate for isoprene a ROS-related function, eventually co-regulating the plant-internal signalling network and development processes in root tissue.
Assuntos
Butadienos/metabolismo , Hemiterpenos/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Populus/crescimento & desenvolvimento , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Arabidopsis/genética , Células Vegetais , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Populus/genética , Populus/metabolismo , Regiões Promotoras Genéticas , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico , Compostos Orgânicos Voláteis/metabolismoRESUMO
Scar formation after brain injury is still poorly understood. To further elucidate such processes, here, we examine the interplay between astrocyte proliferation taking place predominantly at the vascular interface and monocyte invasion. Using genetic mouse models that decrease or increase reactive astrocyte proliferation, we demonstrate inverse effects on monocyte numbers in the injury site. Conversely, reducing monocyte invasion using CCR2-/- mice causes a strong increase in astrocyte proliferation, demonstrating an intriguing negative cross-regulation between these cell types at the vascular interface. CCR2-/- mice show reduced scar formation with less extracellular matrix deposition, smaller lesion site and increased neuronal coverage. Surprisingly, the GFAP+ scar area in these mice is also significantly decreased despite increased astrocyte proliferation. Proteomic analysis at the peak of increased astrocyte proliferation reveals a decrease in extracellular matrix synthesizing enzymes in the injury sites of CCR2-/- mice, highlighting how early key aspects of scar formation are initiated. Taken together, we provide novel insights into the cross-regulation of juxtavascular proliferating astrocytes and invading monocytes as a crucial mechanism of scar formation upon brain injury.
Assuntos
Astrócitos/citologia , Lesões Encefálicas/patologia , Proliferação de Células , Cicatriz/genética , Monócitos/citologia , Transdução de Sinais , Animais , Células Cultivadas , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteômica , Receptores de Hidrocarboneto Arílico/genética , Receptores CCR2/genéticaRESUMO
Epoxyeicosatrienoic acids (EETs) are potent lipid mediators with well-established effects in vascular tissues. Recent studies indicated an emerging role of these eicosanoids in metabolic diseases and the EET signaling pathway was shown to be involved in hepatic insulin sensitivity. However, compared to vascular tissues, there is only limited knowledge about the underlying signaling pathways in the liver. Therefore, we employed an LC-MS/MS-based time-resolved phosphoproteomics approach to characterize 11,12-EET-mediated signaling events in the liver cell line Hepa 1-6. 11,12-EET treatment resulted in the time-dependent regulation of phosphopeptides involved in processes as yet unknown to be affected by EETs, including RNA processing, splicing and translation regulation. Pathway analysis combined with western blot-based validation revealed enhanced AKT/mTOR/p70S6K signaling as demonstrated by increased acute phosphorylation of AKT (Ser473) and p70S6K (Thr389). In addition, 11,12-EET treatment led to differential regulation of phosphopeptides including important mediators of the DNA damage response and we observed a prolonged induction of the etoposide-induced DNA damage marker γH2AX in response to 11,12-EET. In summary, our findings extend current knowledge of 11,12-EET signaling events and emphasize the importance of the AKT/mTOR/p70S6K pathway in hepatic 11,12-EET signaling. Based on the results presented in this study, we furthermore propose a novel role of EET signaling in the regulation of the DNA damage response.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Fígado/metabolismo , Proteômica , Transdução de Sinais , Ácido 8,11,14-Eicosatrienoico/metabolismo , Linhagem Celular , Humanos , FosfoproteínasRESUMO
Normal tissue toxicity is a dose-limiting factor in radiation therapy. Therefore, a detailed understanding of the normal tissue response to radiation is necessary to predict the risk of normal tissue toxicity and to development strategies for tissue protection. One component of normal tissue that is continuously exposed during therapeutic irradiation is the circulating population of peripheral blood mononuclear cells (PBMC). PBMCs are highly sensitive to ionizing radiation (IR); however, little is known about how IR affects the PBMC response on a systemic level. It was the aim of this study to investigate whether IR was capable to induce changes in the composition and function of extracellular vesicles (EVs) secreted from PBMCs after radiation exposure to different doses. Therefore, whole blood samples from healthy donors were exposed to X-ray radiation in the clinically relevant doses of 0, 0.1, 2 or 6 Gy and PBMC-secreted EVs were isolated 72 h later. Proteome and miRNome analysis of EVs as well as functional studies were performed. Secreted EVs showed a dose-dependent increase in the number of significantly deregulated proteins and microRNAs. For both, proteome and microRNA data, principal component analysis showed a dose-dependent separation of control and exposed groups. Integrated pathway analysis of the radiation-regulated EV proteins and microRNAs consistently predicted an association of deregulated molecules with apoptosis, cell death and survival. Functional studies identified endothelial cells as an efficient EV recipient system, in which irradiation of recipient cells further increased the uptake. Furthermore an apoptosis suppressive effect of EVs from irradiated PBMCs in endothelial recipient cells was detected. In summary, this study demonstrates that IR modifies the communication between PBMCs and endothelial cells. EVs from irradiated PBMC donors were identified as transmitters of protective signals to irradiated endothelial cells. Thus, these data may lead to the discovery of biomarker candidates for radiation dosimetry and even more importantly, they suggest EVs as a novel systemic communication pathway between irradiated normal, non-cancer tissues.
Assuntos
Vesículas Extracelulares/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/efeitos da radiação , Exposição à Radiação , Vesículas Secretórias/metabolismo , Apoptose/efeitos da radiação , Células Endoteliais/metabolismo , Células Endoteliais/efeitos da radiação , Humanos , MicroRNAs/genética , Proteoma/metabolismo , Radiação Ionizante , Radioterapia/métodosRESUMO
Jumonji-domain-containing protein 6 (JMJD6) is a Fe(II) and 2-oxogluterate (2OG) dependent oxygenase involved in gene regulation through post-translationally modifying nuclear proteins. It is highly expressed in many cancer types and linked to tumor progression and metastasis. Four alternatively-spliced jmjd6 transcripts were annotated. Here, we focus on the two most abundantly expressed ones, which we call jmjd6-2 and jmjd6-Ex5. TCGA SpliceSeq data revealed a significant decrease of jmjd6-Ex5 transcripts in patients and postmortem tissue of several tumors. The two protein isoforms are distinguished by their C-terminal sequences, which include a serine-rich region (polyS-domain) in JMJD6-2 that is not present in JMJD6-Ex5. Immunoprecipitation followed by LC-MS/MS for JMJD6-Ex5 shows that different sets of proteins interact with JMJD6-2 and JMJD6-Ex5 with only a few overlaps. In particular, we found TFIIF-associating CTD phosphatase (FCP1), proteins of the survival of motor neurons (SMN) complex, heterogeneous nuclear ribonucleoproteins (hnRNPs) and upstream binding factor (UBF) to interact with JMJD6-Ex5. Like JMJD6-2, both UBF and FCP1 comprise a polyS-domain. The polyS domain of JMJD6-2 might block the interaction with polyS-domains of other proteins. In contrast, JMJD6-2 interacts with many SR-like proteins with arginine/serine-rich (RS)-domains, including several splicing factors. In an HIV-based splicing reporter assay, co-expression of JMJD6-2 inhibited exon inclusion, whereas JMJD6-Ex5 did not have any effect. Furthermore, the silencing of jmjd6 by siRNAs favored jmjd6-Ex5 transcripts, suggesting that JMJD6 controls splicing of its own pre-mRNA. The distinct molecular properties of JMJD6-2 and JMJD6-Ex5 open a lead into the functional implications of the variations of their relative abundance in tumors.
Assuntos
Histona Desmetilases com o Domínio Jumonji/metabolismo , Splicing de RNA , Células HEK293 , Células HeLa , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Neoplasias/metabolismo , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMO
Epidemiological studies on workers employed at the Mayak plutonium enrichment plant have demonstrated an association between external gamma ray exposure and an elevated risk of ischemic heart disease (IHD). In a previous study using fresh-frozen post mortem samples of the cardiac left ventricle of Mayak workers and non-irradiated controls, we observed radiation-induced alterations in the heart proteome, mainly downregulation of mitochondrial and structural proteins. As the control group available at that time was younger than the irradiated group, we could not exclude age as a confounding factor. To address this issue, we have now expanded our study to investigate additional samples using archival formalin-fixed paraffin-embedded (FFPE) tissue. Importantly, the control group studied here is older than the occupationally exposed (>500 mGy) group. Label-free quantitative proteomics analysis showed that proteins involved in the lipid metabolism, sirtuin signaling, mitochondrial function, cytoskeletal organization, and antioxidant defense were the most affected. A histopathological analysis elucidated large foci of fibrotic tissue, myocardial lipomatosis and lymphocytic infiltrations in the irradiated samples. These data highlight the suitability of FFPE material for proteomics analysis. The study confirms the previous results emphasizing the role of adverse metabolic changes in the radiation-associated IHD. Most importantly, it excludes age at the time of death as a confounding factor.
Assuntos
Isquemia Miocárdica/metabolismo , Plutônio/efeitos adversos , Proteoma/metabolismo , Proteoma/efeitos da radiação , Cromatografia Líquida , Citoesqueleto/metabolismo , Citoesqueleto/efeitos da radiação , Formaldeído/química , Humanos , Metabolismo dos Lipídeos/efeitos da radiação , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Isquemia Miocárdica/epidemiologia , Isquemia Miocárdica/etiologia , Isquemia Miocárdica/patologia , Exposição Ocupacional , Inclusão em Parafina , Análise de Componente Principal , Mapas de Interação de Proteínas , Proteômica/métodos , Radiação Ionizante , Transdução de Sinais/efeitos da radiação , Sirtuínas/metabolismo , Espectrometria de Massas em Tandem , Fixação de TecidosRESUMO
Identification of interactors is a major goal in cell biology. Not only protein-protein but also protein-carbohydrate interactions are of high relevance for signal transduction in biological systems. Here, we aim to identify novel interacting binding partners for the ß-galactoside-binding proteins galectin-1 (Gal-1) and galectin-3 (Gal-3) relevant in the context of the eye disease proliferative vitreoretinopathy (PVR). PVR is one of the most common failures after retinal detachment surgeries and is characterized by the migration, adhesion, and epithelial-to-mesenchymal transition of retinal pigment epithelial cells (RPE) and the subsequent formation of sub- and epiretinal fibrocellular membranes. Gal-1 and Gal-3 bind in a dose- and carbohydrate-dependent manner to mesenchymal RPE cells and inhibit cellular processes like attachment and spreading. Yet knowledge about glycan-dependent interactors of Gal-1 and Gal-3 on RPE cells is very limited, although this is a prerequisite for unraveling the influence of galectins on distinct cellular processes in RPE cells. We identify here 131 Gal-3 and 15 Gal-1 interactors by galectin pulldown experiments combined with quantitative proteomics. They mainly play a role in multiple binding processes and are mostly membrane proteins. We focused on two novel identified interactors of Gal-1 and Gal-3 in the context of PVR: the low-density lipoprotein receptor LRP1 and the platelet-derived growth factor receptor ß PDGFRB. Addition of exogenous Gal-1 and Gal-3 induced cross-linking with LRP1/PDGFRB and integrin-ß1 (ITGB1) on the cell surface of human RPE cells and induced ERK/MAPK and Akt signaling. Treatment with kifunensine, an inhibitor of complex-type N-glycosylation, weakened the binding of Gal-1 and Gal-3 to these interactors and prevented lattice formation. In conclusion, the identified specific glycoprotein ligands shed light into the highly specific binding of galectins to dedifferentiated RPE cells and the resulting prevention of PVR-associated cellular events.
Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Galectina 1/metabolismo , Galectina 3/metabolismo , Proteoma/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Vitreorretinopatia Proliferativa/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Alcaloides/farmacologia , Proteínas Sanguíneas , Membrana Celular/metabolismo , Células Cultivadas , Citoesqueleto/metabolismo , Inibidores Enzimáticos/farmacologia , Galectinas , Glicosilação/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
Chronic exposure to low-dose ionizing radiation is associated with an increased risk of cardiovascular disease. Alteration in energy metabolism has been suggested to contribute to radiation-induced heart pathology, mitochondrial dysfunction being a hallmark of this disease. The goal of this study was to investigate the regulatory role of acetylation in heart mitochondria in the long-term response to chronic radiation. ApoE-deficient C57Bl/6J mice were exposed to low-dose-rate (20 mGy/day) gamma radiation for 300 days, resulting in a cumulative total body dose of 6.0 Gy. Heart mitochondria were isolated and analyzed using quantitative proteomics. Radiation-induced proteome and acetylome alterations were further validated using immunoblotting, enzyme activity assays, and ELISA. In total, 71 proteins showed peptides with a changed acetylation status following irradiation. The great majority (94%) of the hyperacetylated proteins were involved in the TCA cycle, fatty acid oxidation, oxidative stress response and sirtuin pathway. The elevated acetylation patterns coincided with reduced activity of mitochondrial sirtuins, increased the level of Acetyl-CoA, and were accompanied by inactivation of major cardiac metabolic regulators PGC-1 alpha and PPAR alpha. These observations suggest that the changes in mitochondrial acetylation after irradiation is associated with impairment of heart metabolism. We propose a novel mechanism involved in the development of late cardiac damage following chronic irradiation.