RESUMO
For healthspan and lifespan, ERK, AMPK and mTORC1 represent critical pathways and inflammation is a centrally important hallmark1-7. Here we examined whether IL-11, a pro-inflammatory cytokine of the IL-6 family, has a negative effect on age-associated disease and lifespan. As mice age, IL-11 is upregulated across cell types and tissues to regulate an ERK-AMPK-mTORC1 axis to modulate cellular, tissue- and organismal-level ageing pathologies. Deletion of Il11 or Il11ra1 protects against metabolic decline, multi-morbidity and frailty in old age. Administration of anti-IL-11 to 75-week-old mice for 25 weeks improves metabolism and muscle function, and reduces ageing biomarkers and frailty across sexes. In lifespan studies, genetic deletion of Il11 extended the lives of mice of both sexes, by 24.9% on average. Treatment with anti-IL-11 from 75 weeks of age until death extends the median lifespan of male mice by 22.5% and of female mice by 25%. Together, these results demonstrate a role for the pro-inflammatory factor IL-11 in mammalian healthspan and lifespan. We suggest that anti-IL-11 therapy, which is currently in early-stage clinical trials for fibrotic lung disease, may provide a translational opportunity to determine the effects of IL-11 inhibition on ageing pathologies in older people.
Assuntos
Envelhecimento , Interleucina-11 , Longevidade , Transdução de Sinais , Animais , Feminino , Masculino , Camundongos , Envelhecimento/efeitos dos fármacos , Envelhecimento/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Proteínas Quinases Ativadas por AMP/metabolismo , Fragilidade/genética , Fragilidade/metabolismo , Fragilidade/prevenção & controle , Inflamação/metabolismo , Inflamação/tratamento farmacológico , Interleucina-11/antagonistas & inibidores , Interleucina-11/deficiência , Interleucina-11/genética , Interleucina-11/metabolismo , Subunidade alfa de Receptor de Interleucina-11/metabolismo , Subunidade alfa de Receptor de Interleucina-11/deficiência , Longevidade/efeitos dos fármacos , Longevidade/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Humanos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologiaRESUMO
Translated small open reading frames (smORFs) can have important regulatory roles and encode microproteins, yet their genome-wide identification has been challenging. We determined the ribosome locations across six primary human cell types and five tissues and detected 7,767 smORFs with translational profiles matching those of known proteins. The human genome was found to contain highly cell-type- and tissue-specific smORFs and a subset that encodes highly conserved amino acid sequences. Changes in the translational efficiency of upstream-encoded smORFs (uORFs) and the corresponding main ORFs predominantly occur in the same direction. Integration with 456 mass-spectrometry datasets confirms the presence of 603 small peptides at the protein level in humans and provides insights into the subcellular localization of these small proteins. This study provides a comprehensive atlas of high-confidence translated smORFs derived from primary human cells and tissues in order to provide a more complete understanding of the translated human genome.
Assuntos
Regulação da Expressão Gênica , Ribossomos , Genoma Humano/genética , Humanos , Fases de Leitura Aberta/genética , Biossíntese de Proteínas , Proteínas/metabolismo , RNA/metabolismo , Ribossomos/genética , Ribossomos/metabolismoRESUMO
The lysosome is central to the degradation of proteins, carbohydrates, and lipids and their salvage back to the cytosol for reutilization. Lysosomal transporters for amino acids, sugars, and cholesterol have been identified, and the metabolic fates of these molecules in the cytoplasm have been elucidated. Remarkably, it is not known whether lysosomal salvage exists for glycerophospholipids, the major constituents of cellular membranes. By using a transport assay screen against orphan lysosomal transporters, we identified the major facilitator superfamily protein Spns1 that is ubiquitously expressed in all tissues as a proton-dependent lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) transporter, with LPC and LPE being the lysosomal breakdown products of the most abundant eukaryotic phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively. Spns1 deficiency in cells, zebrafish embryos, and mouse liver resulted in lysosomal accumulation of LPC and LPE species with pathological consequences on lysosomal function. Flux analysis using stable isotope-labeled phospholipid apolipoprotein E nanodiscs targeted to lysosomes showed that LPC was transported out of lysosomes in an Spns1-dependent manner and re-esterified back into the cytoplasmic pools of phosphatidylcholine. Our findings identify a phospholipid salvage pathway from lysosomes to the cytosol that is dependent on Spns1 and critical for maintaining normal lysosomal function.
Assuntos
Lisofosfolipídeos , Proteínas de Membrana Transportadoras , Fosfatidiletanolaminas , Peixe-Zebra , Animais , Lisofosfatidilcolinas/metabolismo , Lisofosfolipídeos/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Prótons , Peixe-Zebra/metabolismo , Proteínas de Peixe-ZebraRESUMO
MOTIVATION: The creation and analysis of gene regulatory networks have been the focus of bioinformatics research and underpins much of what is known about gene regulation. However, as a result of a bias in the availability of data types that are collected, the vast majority of gene regulatory network resources and tools have focused on either transcriptional regulation or protein-protein interactions. This has left other areas of regulation, for instance, translational regulation, vastly underrepresented despite them having been shown to play a critical role in both health and disease. RESULTS: In order to address this, we have developed CLIPreg, a package that integrates RNA, Ribo and CLIP- sequencing data in order to construct translational regulatory networks coordinated by RNA-binding proteins and micro-RNAs. This is the first tool of its type to be created, allowing for detailed investigation into a previously unseen layer of regulation. AVAILABILITY AND IMPLEMENTATION: CLIPreg is available at https://github.com/SGDDNB/CLIPreg. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Redes Reguladoras de Genes , MicroRNAs , RNA-Seq , Proteínas de Ligação a RNA , SoftwareRESUMO
Building a reference set of protein-coding open reading frames (ORFs) has revolutionized biological process discovery and understanding. Traditionally, gene models have been confirmed using cDNA sequencing and encoded translated regions inferred using sequence-based detection of start and stop combinations longer than 100 amino-acids to prevent false positives. This has led to small ORFs (smORFs) and their encoded proteins left un-annotated. Ribo-seq allows deciphering translated regions from untranslated irrespective of the length. In this review, we describe the power of Ribo-seq data in detection of smORFs while discussing the major challenge posed by data-quality, -depth and -sparseness in identifying the start and end of smORF translation. In particular, we outline smORF cataloguing efforts in humans and the large differences that have arisen due to variation in data, methods and assumptions. Although current versions of smORF reference sets can already be used as a powerful tool for hypothesis generation, we recommend that future editions should consider these data limitations and adopt unified processing for the community to establish a canonical catalogue of translated smORFs.
Assuntos
Proteínas , Perfil de Ribossomos , Humanos , Proteínas/genética , Fases de Leitura Aberta , Biossíntese de Proteínas , MicropeptídeosRESUMO
The interplay between different levels of gene regulation in modulating developmental transcriptional programs, such as histone modifications and chromatin remodeling, is not well understood. Here, we show that the chromatin remodeling factor Brg1 is required for enhancer activation in mesoderm induction. In an embryonic stem cell-based directed differentiation assay, the absence of Brg1 results in a failure of cardiomyocyte differentiation and broad deregulation of lineage-specific gene expression during mesoderm induction. We find that Brg1 co-localizes with H3K27ac at distal enhancers and is required for robust H3K27 acetylation at distal enhancers that are activated during mesoderm induction. Brg1 is also required to maintain Polycomb-mediated repression of non-mesodermal developmental regulators, suggesting cooperativity between Brg1 and Polycomb complexes. Thus, Brg1 is essential for modulating active and repressive chromatin states during mesoderm lineage commitment, in particular the activation of developmentally important enhancers. These findings demonstrate interplay between chromatin remodeling complexes and histone modifications that, together, ensure robust and broad gene regulation during crucial lineage commitment decisions.
Assuntos
DNA Helicases/metabolismo , Elementos Facilitadores Genéticos/fisiologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem da Célula , Cromatina/metabolismo , DNA Helicases/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos/genética , Inativação Gênica/fisiologia , Histonas/metabolismo , Humanos , Mesoderma/citologia , Mesoderma/metabolismo , Mutação , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteínas Nucleares/genética , Proteínas do Grupo Polycomb/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Fatores de Transcrição/genéticaRESUMO
In mammals, combinatorial assembly of alternative families of subunits confers functional specificity to adenosine triphosphate (ATP)-dependent SWI/SNF-like Brg/Brm-associated factor (BAF) chromatin remodeling complexes by creating distinct polymorphic surfaces for interaction with regulatory elements and DNA-binding factors. Although redundant in terms of biochemical activity, the core ATPase subunits, BRG/SMARCA4 and BRM/SMARCA2, are functionally distinct and may contribute to complex specificity. Here we show using quantitative proteomics that BAF complexes expressed in leukemia are specifically assembled around the BRG ATPase. Moreover, using a mouse model of acute myeloid leukemia, we demonstrate that BRG is essential for leukemia maintenance, as leukemic cells lacking BRG rapidly undergo cell-cycle arrest and apoptosis. Most importantly, we show that BRG is dispensable for the maintenance of immunophenotypic long-term repopulating hematopoietic stem cells, suggesting that adroit targeting of BRG in leukemia may have potent and specific therapeutic effects.
Assuntos
Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , DNA Helicases/fisiologia , Células-Tronco Hematopoéticas/patologia , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Animais , Southern Blotting , Cromatografia Líquida , DNA Helicases/genética , Modelos Animais de Doenças , Citometria de Fluxo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem , Fatores de Transcrição/genéticaRESUMO
New methods for the genome-wide analysis of chromatin are providing insight into its roles in development and their underlying mechanisms. Current studies indicate that chromatin is dynamic, with its structure and its histone modifications undergoing global changes during transitions in development and in response to extracellular cues. In addition to DNA methylation and histone modification, ATP-dependent enzymes that remodel chromatin are important controllers of chromatin structure and assembly, and are major contributors to the dynamic nature of chromatin. Evidence is emerging that these chromatin-remodelling enzymes have instructive and programmatic roles during development. Particularly intriguing are the findings that specialized assemblies of ATP-dependent remodellers are essential for establishing and maintaining pluripotent and multipotent states in cells.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/enzimologia , Animais , Padronização Corporal/genética , DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Conformação de Ácido Nucleico , Fatores de Transcrição/metabolismoRESUMO
Epigenetic modifications must underlie lineage-specific differentiation as terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Haematopoiesis provides a well-defined model to study epigenetic modifications during cell-fate decisions, as multipotent progenitors (MPPs) differentiate into progressively restricted myeloid or lymphoid progenitors. Although DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs, a comprehensive DNA methylation map of haematopoietic progenitors, or of any multipotent/oligopotent lineage, does not exist. Here we examined 4.6 million CpG sites throughout the genome for MPPs, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and thymocyte progenitors (DN1, DN2, DN3). Marked epigenetic plasticity accompanied both lymphoid and myeloid restriction. Myeloid commitment involved less global DNA methylation than lymphoid commitment, supported functionally by myeloid skewing of progenitors following treatment with a DNA methyltransferase inhibitor. Differential DNA methylation correlated with gene expression more strongly at CpG island shores than CpG islands. Many examples of genes and pathways not previously known to be involved in choice between lymphoid/myeloid differentiation have been identified, such as Arl4c and Jdp2. Several transcription factors, including Meis1, were methylated and silenced during differentiation, indicating a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome seems to be important in haematopoietic differentiation. Our results directly demonstrate that modulation of DNA methylation occurs during lineage-specific differentiation and defines a comprehensive map of the methylation and transcriptional changes that accompany myeloid versus lymphoid fate decisions.
Assuntos
Linhagem da Célula , Metilação de DNA , Hematopoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Animais , Linhagem Celular , Linhagem da Célula/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Epigênese Genética , Perfilação da Expressão Gênica , Genoma/genética , Hematopoese/genética , Linfócitos/citologia , Linfócitos/metabolismo , Metaboloma , Metabolômica , Camundongos , Células Mieloides/citologia , Células Mieloides/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismoRESUMO
Oral-facial-digital syndrome type VI (OFD VI) is a recessive ciliopathy defined by two diagnostic criteria: molar tooth sign (MTS) and one or more of the following: (1) tongue hamartoma (s) and/or additional frenula and/or upper lip notch; (2) mesoaxial polydactyly of one or more hands or feet; (3) hypothalamic hamartoma. Because of the MTS, OFD VI belongs to the "Joubert syndrome related disorders". Its genetic aetiology remains largely unknown although mutations in the TMEM216 gene, responsible for Joubert (JBS2) and Meckel-Gruber (MKS2) syndromes, have been reported in two OFD VI patients. To explore the molecular cause(s) of OFD VI syndrome, we used an exome sequencing strategy in six unrelated families followed by Sanger sequencing. We identified a total of 14 novel mutations in the C5orf42 gene in 9/11 families with positive OFD VI diagnostic criteria including a severe fetal case with microphthalmia, cerebellar hypoplasia, corpus callosum agenesis, polydactyly and skeletal dysplasia. C5orf42 mutations have already been reported in Joubert syndrome confirming that OFD VI and JBS are allelic disorders, thus enhancing our knowledge of the complex, highly heterogeneous nature of ciliopathies.
Assuntos
Proteínas de Membrana/genética , Síndromes Orofaciodigitais/diagnóstico , Síndromes Orofaciodigitais/genética , Anormalidades Múltiplas , Adolescente , Adulto , Alelos , Doenças Cerebelares/diagnóstico , Doenças Cerebelares/genética , Cerebelo/anormalidades , Criança , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/genética , Exoma , Anormalidades do Olho/diagnóstico , Anormalidades do Olho/genética , Feminino , Hamartoma/diagnóstico , Hamartoma/genética , Humanos , Doenças Hipotalâmicas/diagnóstico , Doenças Hipotalâmicas/genética , Doenças Renais Císticas/diagnóstico , Doenças Renais Císticas/genética , Masculino , Mutação , Malformações do Sistema Nervoso/diagnóstico , Malformações do Sistema Nervoso/genética , Fenótipo , Polidactilia/diagnóstico , Polidactilia/genética , Retina/anormalidades , Análise de Sequência de DNA , Adulto JovemRESUMO
Protein translation is an energy-intensive ribosome-driven process that is reduced during nutrient scarcity to conserve cellular resources. During prolonged starvation, cells selectively translate specific proteins to enhance their survival (adaptive translation); however, this process is poorly understood. Accordingly, we analyzed protein translation and mRNA transcription by multiple methods in vitro and in vivo to investigate adaptive hepatic translation during starvation. While acute starvation suppressed protein translation in general, proteomic analysis showed that prolonged starvation selectively induced translation of lysosome and autolysosome proteins. Significantly, the expression of the orphan nuclear receptor, estrogen-related receptor alpha (Esrra) increased during prolonged starvation and served as a master regulator of this adaptive translation by transcriptionally stimulating 60S acidic ribosomal protein P1 (Rplp1) gene expression. Overexpression or siRNA knockdown of Esrra expression in vitro or in vivo led to parallel changes in Rplp1 gene expression, lysosome/autophagy protein translation, and autophagy. Remarkably, we have found that Esrra had dual functions by not only regulating transcription but also controling adaptive translation via the Esrra/Rplp1/lysosome/autophagy pathway during prolonged starvation.
RESUMO
At critical times in development, cells are able to convert graded signals into discrete developmental outcomes; however, the mechanisms involved are poorly understood. During thymocyte development, cell fate is determined by signals originating from the alphabeta T-cell receptor. Low-affinity/avidity interactions between the T-cell receptor and peptide-MHC complexes direct differentiation to the single-positive stage (positive selection), whereas high-affinity/avidity interactions induce death by apoptosis (negative selection). Here we show that mice deficient in both calcineurin and nuclear factor of activated T cells (NFAT)c2/c3 lack a population of preselection thymocytes with enhanced ability to activate the mitogen-activated protein kinase (Raf-MEK-ERK) pathway, and fail to undergo positive selection. This defect can be partially rescued with constitutively active Raf, indicating that calcineurin controls MAPK signalling. Analysis of mice deficient in both Bim (which is required for negative selection) and calcineurin revealed that calcineurin-induced ERK (extracellular signal-regulated kinase) sensitization is required for differentiation in response to 'weak' positive selecting signals but not in response to 'strong' negative selecting signals (which normally induce apoptosis). These results indicate that early calcineurin/NFAT signalling produces a developmental period of ERK hypersensitivity, allowing very weak signals to induce positive selection. This mechanism might be generally useful in the discrimination of graded signals that induce different cell fates.
Assuntos
Calcineurina/metabolismo , Diferenciação Celular , Sistema de Sinalização das MAP Quinases , Timo/citologia , Timo/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Calcineurina/deficiência , Calcineurina/genética , Células Cultivadas , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ligantes , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fatores de Transcrição NFATC/metabolismo , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Timo/enzimologia , Quinases raf/metabolismoRESUMO
LINC00116 encodes a microprotein first identified as Mitoregulin (MTLN), where it was reported to localize to the inner membrane of mitochondria to regulate fatty acid oxidation and oxidative phosphorylation. These initial discoveries were followed by reports with differing findings about its molecular functions and submitochondrial localization. To clarify the apparent discrepancies, we constructed multiple orthogonal methods of determining the localization of MTLN, including split GFP-based reporters that enable efficient and reliable topology analyses for microproteins. These methods unequivocally demonstrate MTLN primarily localizes to the outer membrane of mitochondria, where it interacts with enzymes of fatty acid metabolism including CPT1B and CYB5B. Loss of MTLN causes the accumulation of very long-chain fatty acids (VLCFAs), especially docosahexaenoic acid (DHA). Intriguingly, loss of MTLN protects mice against western diet/fructose-induced insulin-resistance, suggests a protective effect of VLCFAs in this context. MTLN thus serves as an attractive target to control the catabolism of VLCFAs.
RESUMO
Lung inflammation is a hallmark of Coronavirus disease 2019 (COVID-19) in patients who are severely ill, and the pathophysiology of disease is thought to be immune mediated. Mast cells (MCs) are polyfunctional immune cells present in the airways, where they respond to certain viruses and allergens and often promote inflammation. We observed widespread degranulation of MCs during acute and unresolved airway inflammation in SARS-CoV-2-infected mice and nonhuman primates. Using a mouse model of MC deficiency, MC-dependent interstitial pneumonitis, hemorrhaging, and edema in the lung were observed during SARS-CoV-2 infection. In humans, transcriptional changes in patients requiring oxygen supplementation also implicated cells with a MC phenotype in severe disease. MC activation in humans was confirmed through detection of MC-specific proteases, including chymase, the levels of which were significantly correlated with disease severity and with biomarkers of vascular dysregulation. These results support the involvement of MCs in lung tissue damage during SARS-CoV-2 infection in animal models and the association of MC activation with severe COVID-19 in humans, suggesting potential strategies for intervention.
Assuntos
COVID-19 , Humanos , Animais , COVID-19/patologia , Mastócitos/patologia , SARS-CoV-2 , Pulmão/patologia , Inflamação/patologiaRESUMO
Recognition of intracellular bacteria by macrophages leads to secretion of type I IFNs. However, the role of type I IFN during bacterial infection is still poorly understood. Francisella tularensis, the causative agent of tularemia, is a pathogenic bacterium that replicates in the cytosol of macrophages leading to secretion of type I IFN. In this study, we investigated the role of type I IFNs in a mouse model of tularemia. Mice deficient for type I IFN receptor (IFNAR1(-/-)) are more resistant to intradermal infection with F. tularensis subspecies novicida (F. novicida). Increased resistance to infection was associated with a specific increase in IL-17A/F and a corresponding expansion of an IL-17A(+) gammadelta T cell population, indicating that type I IFNs negatively regulate the number of IL-17A(+) gammadelta T cells during infection. Furthermore, IL-17A-deficient mice contained fewer neutrophils compared with wild-type mice during infection, indicating that IL-17A contributes to neutrophil expansion during F. novicida infection. Accordingly, an increase in IL-17A in IFNAR1(-/-) mice correlated with an increase in splenic neutrophil numbers. Similar results were obtained in a mouse model of pneumonic tularemia using the highly virulent F. tularensis subspecies tularensis SchuS4 strain and in a mouse model of systemic Listeria monocytogenes infection. Our results indicate that the type I IFN-mediated negative regulation of IL-17A(+) gammadelta T cell expansion is conserved during bacterial infections. We propose that this newly described activity of type I IFN signaling might participate in the resistance of the IFNAR1(-/-) mice to infection with F. novicida and other intracellular bacteria.
Assuntos
Infecções Bacterianas/imunologia , Interferon Tipo I/imunologia , Interleucina-17/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Animais , Infecções Bacterianas/metabolismo , Separação Celular , Citometria de Fluxo , Interleucina-17/imunologia , Listeriose/imunologia , Listeriose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , Tularemia/imunologia , Tularemia/metabolismoRESUMO
Distinctive SWI/SNF-like ATP-dependent chromatin remodeling esBAF complexes are indispensable for the maintenance and pluripotency of mouse embryonic stem (ES) cells [Ho L, et al. (2009) Proc Natl Acad Sci USA 10.1073/pnas.0812889106]. To understand the mechanism underlying the roles of these complexes in ES cells, we performed high-resolution genome-wide mapping of the core ATPase subunit, Brg, using ChIP-Seq technology. We find that esBAF, as represented by Brg, binds to genes encoding components of the core ES transcriptional circuitry, including Polycomb group proteins. esBAF colocalizes extensively with transcription factors Oct4, Sox2 and Nanog genome-wide, and shows distinct functional interactions with Oct4 and Sox2 at its target genes. Surprisingly, no significant colocalization of esBAF with PRC2 complexes, represented by Suz12, is observed. Lastly, esBAF colocalizes with Stat3 and Smad1 genome-wide, consistent with a direct and critical role in LIF and BMP signaling for maintaining self-renewal. Taken together, our studies indicate that esBAF is an essential component of the core pluripotency transcriptional network, and might also be a critical component of the LIF and BMP signaling pathways essential for maintenance of self-renewal and pluripotency.
Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Células-Tronco Embrionárias/citologia , Redes Reguladoras de Genes , Células-Tronco Pluripotentes , Fatores de Transcrição/fisiologia , Adenosina Trifosfatases , Animais , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Genômica , Camundongos , Ligação Proteica , Receptores de OSM-LIF/metabolismo , Transdução de SinaisRESUMO
Mammalian SWI/SNF [also called BAF (Brg/Brahma-associated factors)] ATP-dependent chromatin remodeling complexes are essential for formation of the totipotent and pluripotent cells of the early embryo. In addition, subunits of this complex have been recovered in screens for genes required for nuclear reprogramming in Xenopus and mouse embryonic stem cell (ES) morphology. However, the mechanism underlying the roles of these complexes is unclear. Here, we show that BAF complexes are required for the self-renewal and pluripotency of mouse ES cells but not for the proliferation of fibroblasts or other cells. Proteomic studies reveal that ES cells express distinctive complexes (esBAF) defined by the presence of Brg (Brahma-related gene), BAF155, and BAF60A, and the absence of Brm (Brahma), BAF170, and BAF60C. We show that this specialized subunit composition is required for ES cell maintenance and pluripotency. Our proteomic analysis also reveals that esBAF complexes interact directly with key regulators of pluripotency, suggesting that esBAF complexes are specialized to interact with ES cell-specific regulators, providing a potential explanation for the requirement of BAF complexes in pluripotency.
Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/fisiologia , Animais , Proliferação de Células , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/análise , Fibroblastos/citologia , Camundongos , Proteínas Musculares/análise , Proteômica , Fatores de Transcrição/análiseRESUMO
Nucleotide-binding oligomerization domain (NBD), leucine-rich repeat (LRR) containing protein family (NLRs) are intracellular pattern recognition receptors that mediate innate immunity against infections. The endothelium is the first line of defense against blood-borne pathogens, but it is unclear which NLRs control endothelial cell (EC) intrinsic immunity. Here, we demonstrate that human ECs simultaneously activate NLRP1 and CARD8 inflammasomes in response to DPP8/9 inhibitor Val-boro-Pro (VbP). Enterovirus Coxsackie virus B3 (CVB3)-the most common cause of viral myocarditis-predominantly activates CARD8 in ECs in a manner that requires viral 2A and 3C protease cleavage at CARD8 p.G38 and proteasome function. Genetic deletion of CARD8 in ECs and human embryonic stem cell-derived cardiomyocytes (HCMs) attenuates CVB3-induced pyroptosis, inflammation, and viral propagation. Furthermore, using a stratified endothelial-cardiomyocyte co-culture system, we demonstrate that deleting CARD8 in ECs reduces CVB3 infection of the underlying cardiomyocytes. Our study uncovers the unique role of CARD8 inflammasome in endothelium-intrinsic anti-viral immunity.
Assuntos
Sistema Cardiovascular , Inflamassomos , Proteínas Reguladoras de Apoptose/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Sistema Cardiovascular/metabolismo , Humanos , Inflamassomos/metabolismo , Leucina , Proteínas de Neoplasias/metabolismo , Nucleotídeos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteases ViraisRESUMO
Electron transport chain (ETC) biogenesis is tightly coupled to energy levels and availability of ETC subunits. Complex III (CIII), controlling ubiquinol:ubiquinone ratio in ETC, is an attractive node for modulating ETC levels during metabolic stress. Here, we report the discovery of mammalian Co-ordinator of mitochondrial CYTB (COM) complexes that regulate the stepwise CIII biogenesis in response to nutrient and nuclear-encoded ETC subunit availability. The COMA complex, consisting of UQCC1/2 and membrane anchor C16ORF91, facilitates translation of CIII enzymatic core subunit CYTB. Subsequently, microproteins SMIM4 and BRAWNIN together with COMA subunits form the COMB complex to stabilize nascent CYTB. Finally, UQCC3-containing COMC facilitates CYTB hemylation and association with downstream CIII subunits. Furthermore, when nuclear CIII subunits are limiting, COMB is required to chaperone nascent CYTB to prevent OXPHOS collapse. Our studies highlight CYTB synthesis as a key regulatory node of ETC biogenesis and uncover the roles of microproteins in maintaining mitochondrial homeostasis.
Assuntos
Sinais (Psicologia) , Mitocôndrias , Animais , Transporte de Elétrons , Mamíferos/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismoRESUMO
Human NLRP1 (NACHT, LRR, and PYD domain-containing protein 1) is an innate immune sensor predominantly expressed in the skin and airway epithelium. Here, we report that human NLRP1 senses the ultraviolet B (UVB)- and toxin-induced ribotoxic stress response (RSR). Biochemically, RSR leads to the direct hyperphosphorylation of a human-specific disordered linker region of NLRP1 (NLRP1DR) by MAP3K20/ZAKα kinase and its downstream effector, p38. Mutating a single ZAKα phosphorylation site in NLRP1DR abrogates UVB- and ribotoxin-driven pyroptosis in human keratinocytes. Moreover, fusing NLRP1DR to CARD8, which is insensitive to RSR by itself, creates a minimal inflammasome sensor for UVB and ribotoxins. These results provide insight into UVB sensing by human skin keratinocytes, identify several ribotoxins as NLRP1 agonists, and establish inflammasome-driven pyroptosis as an integral component of the RSR.