RESUMEN
Protein synthesis is a fundamental step in gene expression, with modulation of mRNA translation at the elongation step emerging as an important regulatory node in shaping cellular proteomes. In this context, five distinct lysine methylation events on eukaryotic elongation factor 1A (eEF1A), a fundamental nonribosomal elongation factor, are proposed to influence mRNA translation elongation dynamics. However, a lack of affinity tools has hindered progress in fully understanding how eEF1A lysine methylation impacts protein synthesis. Here we develop and characterize a suite of selective antibodies to investigate eEF1A methylation and provide evidence that methylation levels decline in aged tissue. Determination of the methyl state and stoichiometry on eEF1A in various cell lines by mass spectrometry shows modest cell-to-cell variability. We also find by Western blot analysis that knockdown of individual eEF1A-specific lysine methyltransferases leads to depletion of the cognate lysine methylation event and indicates active crosstalk between different sites. Further, we find that the antibodies are specific in immunohistochemistry applications. Finally, application of the antibody toolkit suggests that several eEF1A methylation events decrease in aged muscle tissue. Together, our study provides a roadmap for leveraging methyl state and sequence-selective antibody reagents to accelerate discovery of eEF1A methylation-related functions and suggests a role for eEF1A methylation, via protein synthesis regulation, in aging biology.
Asunto(s)
Lisina , Extensión de la Cadena Peptídica de Translación , Factor 1 de Elongación Peptídica , Anticuerpos/metabolismo , Lisina/metabolismo , Metilación , Factor 1 de Elongación Peptídica/genética , Factor 1 de Elongación Peptídica/química , Factor 1 de Elongación Peptídica/metabolismoRESUMEN
Although functional association between Wnt signaling and bone homeostasis has been well described through genetic ablation of Wntless (Wls), the mechanisms of how osteoblastic Wls regulates the fate of bone marrow stromal cells (BMSCs) and hematopoietic stem cells (HSCs) in relation to age are not yet understood. Here, we generated Col2.3-Cre;Wlsfl/fl mice that were free from premature lethality and investigated age-related impacts of osteoblastic Wls deficiency on hematopoiesis, BM microenvironment, and maintenance of BMSCs (also known as BM-derived mesenchymal stem/stromal cells) and HSCs. Ablation of osteoblastic Wls deteriorated BM microenvironment and bone mass accrual along with age-independent effects on functions of BMSCs. Osteoblastic Wls deletion impaired HSC repopulation and progeny with skewing toward myeloid lineage cells only at old stage. As proven by hallmarks of stem cell senescence, osteoblastic Wls ablation differentially induced senescence of BMSCs and HSCs in relation to age without alteration in their BM frequency. Our findings support that deletion of Wls in Col2.3-expressing cells induces senescence of BMSCs and impairs BM microenvironment in age-independent manner. Overall, long-term deterioration in BM microenvironment contributes to age-related HSC senescence with impaired progeny and hematopoiesis, which also suggests possible roles of osteoblastic Wls on the maintenance of BM HSCs.
Asunto(s)
Envejecimiento/metabolismo , Células de la Médula Ósea/metabolismo , Eliminación de Gen , Osteoblastos/metabolismo , Receptores Acoplados a Proteínas G/deficiencia , Células Madre/metabolismo , Animales , Ratones , Ratones Transgénicos , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
The mitochondrial ADP/ATP carrier (AAC) is a major transport protein of the inner mitochondrial membrane. It exchanges mitochondrial ATP for cytosolic ADP and controls cellular production of ATP. In addition, it has been proposed that AAC mediates mitochondrial uncoupling, but it has proven difficult to demonstrate this function or to elucidate its mechanisms. Here we record AAC currents directly from inner mitochondrial membranes from various mouse tissues and identify two distinct transport modes: ADP/ATP exchange and H+ transport. The AAC-mediated H+ current requires free fatty acids and resembles the H+ leak via the thermogenic uncoupling protein 1 found in brown fat. The ADP/ATP exchange via AAC negatively regulates the H+ leak, but does not completely inhibit it. This suggests that the H+ leak and mitochondrial uncoupling could be dynamically controlled by cellular ATP demand and the rate of ADP/ATP exchange. By mediating two distinct transport modes, ADP/ATP exchange and H+ leak, AAC connects coupled (ATP production) and uncoupled (thermogenesis) energy conversion in mitochondria.
Asunto(s)
Mitocondrias/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , Protones , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Coenzimas/metabolismo , Ácidos Grasos/metabolismo , Transporte Iónico , Masculino , Ratones , Consumo de OxígenoRESUMEN
Coordinated activation of muscle stem cells (known as satellite cells) is critical for postnatal muscle growth and regeneration. The muscle stem cell niche is central for regulating the activation state of satellite cells, but the specific extracellular signals that coordinate this regulation are poorly understood. Here we show that macrophages at sites of muscle injury induce activation of satellite cells via expression of Adamts1. Overexpression of Adamts1 in macrophages in vivo is sufficient to increase satellite cell activation and improve muscle regeneration in young mice. We demonstrate that NOTCH1 is a target of ADAMTS1 metalloproteinase activity, which reduces Notch signaling, leading to increased satellite cell activation. These results identify Adamts1 as a potent extracellular regulator of satellite cell activation and have significant implications for understanding the regulation of satellite cell activity and regeneration after muscle injury.Satellite cells are crucial for growth and regeneration of skeletal muscle. Here the authors show that in response to muscle injury, macrophages secrete Adamts1, which induces satellite cell activation by modulating Notch1 signaling.
Asunto(s)
Proteína ADAMTS1/genética , Macrófagos/metabolismo , Receptor Notch1/genética , Células Satélite del Músculo Esquelético/metabolismo , Proteína ADAMTS1/metabolismo , Animales , Células Cultivadas , Células HEK293 , Humanos , Ratones Transgénicos , Desarrollo de Músculos/genética , Músculo Esquelético/lesiones , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Mutación , Receptor Notch1/metabolismo , Regeneración/genética , Transducción de Señal/genéticaRESUMEN
With aging, there is progressive loss of tissue homeostasis and functional reserve, leading to an impaired response to stress and an increased risk of morbidity and mortality. A key mediator of the cellular response to damage and stress is the transcription factor NF-κB. We demonstrated previously that NF-κB transcriptional activity is upregulated in tissues from both natural aged mice and in a mouse model of a human progeroid syndrome caused by defective repair of DNA damage (ERCC1-deficient mice). We also demonstrated that genetic reduction in the level of the NF-κB subunit p65(RelA) in the Ercc1-/∆ progeroid mouse model of accelerated aging delayed the onset of age-related pathology including muscle wasting, osteoporosis, and intervertebral disk degeneration. Here, we report that the largest fraction of NF-κB -expressing cells in the bone marrow (BM) of aged (>2 year old) mice (C57BL/6-NF-κBEGFP reporter mice) are Gr-1+ CD11b+ myeloid-derived suppressor cells (MDSCs). There was a significant increase in the overall percentage of MDSC present in the BM of aged animals compared with young, a trend also observed in the spleen. However, the function of these cells appears not to be compromised in aged mice. A similar increase of MDSC was observed in BM of progeroid Ercc1-/∆ and BubR1H/H mice. The increase in MDSC in Ercc1-/∆ mice was abrogated by heterozygosity in the p65/RelA subunit of NF-κB. These results suggest that NF-κB activation with aging, at least in part, drives an increase in the percentage of MDSCs, a cell type able to suppress immune cell responses.
Asunto(s)
Envejecimiento/metabolismo , Células de la Médula Ósea/metabolismo , Proliferación Celular/genética , Células Supresoras de Origen Mieloide/metabolismo , Factor de Transcripción ReIA/genética , Envejecimiento/genética , Animales , Células de la Médula Ósea/citología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Endonucleasas/deficiencia , Endonucleasas/genética , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Supresoras de Origen Mieloide/citología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Bazo/citología , Bazo/metabolismo , Factor de Transcripción ReIA/metabolismo , Transcripción GenéticaRESUMEN
Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disorder in obese individuals. Adenine nucleotide translocase (ANT) exchanges ADP/ATP through the mitochondrial inner membrane, and Ant2 is the predominant isoform expressed in the liver. Here we demonstrate that targeted disruption of Ant2 in mouse liver enhances uncoupled respiration without damaging mitochondrial integrity and liver functions. Interestingly, liver specific Ant2 knockout mice are leaner and resistant to hepatic steatosis, obesity and insulin resistance under a lipogenic diet. Protection against fatty liver is partially recapitulated by the systemic administration of low-dose carboxyatractyloside, a specific inhibitor of ANT. Targeted manipulation of hepatic mitochondrial metabolism, particularly through inhibition of ANT, may represent an alternative approach in NAFLD and obesity treatment.
Asunto(s)
Translocador 2 del Nucleótido Adenina/metabolismo , Adenosina Trifosfato/metabolismo , Hígado Graso/metabolismo , Resistencia a la Insulina , Mitocondrias Hepáticas/metabolismo , Sustancias Protectoras/metabolismo , Translocador 2 del Nucleótido Adenina/genética , Animales , Atractilósido/análogos & derivados , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Hígado Graso/terapia , Femenino , Técnica de Clampeo de la Glucosa , Hiperinsulinismo , Metabolismo de los Lípidos , Lipogénesis , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Noqueados , Membranas Mitocondriales/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Enfermedad del Hígado Graso no Alcohólico/terapia , Obesidad/metabolismo , Obesidad/terapia , Ácido Pirúvico/metabolismoRESUMEN
[This corrects the article DOI: 10.1038/ncomms14477.].
RESUMEN
Trithorax proteins and long-intergenic noncoding RNAs are critical regulators of embryonic stem cell pluripotency; however, how they cooperatively regulate germ layer mesoderm specification remains elusive. We report here that HoxBlinc RNA first specifies Flk1(+) mesoderm and then promotes hematopoietic differentiation through regulation of hoxb pathways. HoxBlinc binds to the hoxb genes, recruits Setd1a/MLL1 complexes, and mediates long-range chromatin interactions to activate transcription of the hoxb genes. Depletion of HoxBlinc by shRNA-mediated knockdown or CRISPR-Cas9-mediated genetic deletion inhibits expression of hoxb genes and other factors regulating cardiac/hematopoietic differentiation. Reduced hoxb expression is accompanied by decreased recruitment of Set1/MLL1 and H3K4me3 modification, as well as by reduced chromatin loop formation. Re-expression of hoxb2-b4 genes in HoxBlinc-depleted embryoid bodies rescues Flk1(+) precursors that undergo hematopoietic differentiation. Thus, HoxBlinc plays an important role in controlling hoxb transcription networks that mediate specification of mesoderm-derived Flk1(+) precursors and differentiation of Flk1(+) cells into hematopoietic lineages.
Asunto(s)
Linaje de la Célula/fisiología , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de Homeodominio/biosíntesis , Mesodermo/embriología , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , ARN Largo no Codificante/biosíntesis , Animales , Línea Celular , Embrión de Mamíferos/citología , N-Metiltransferasa de Histona-Lisina/genética , Proteínas de Homeodominio/genética , Mesodermo/citología , Ratones , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/genética , ARN Largo no Codificante/genéticaRESUMEN
Purinergic receptors of the P2Y family are G protein-coupled surface receptors that respond to extracellular nucleotides and can mediate responses to local cell damage. P2Y-dependent signaling contributes to thrombotic and/or inflammatory consequences of tissue injury by altering platelet and endothelial activation and immune cell phagocytosis. Here, we have demonstrated that P2Y14 modifies cell senescence and cell death in response to tissue stress, thereby enabling preservation of hematopoietic stem/progenitor cell function. In mice, P2Y14 deficiency had no demonstrable effect under homeostatic conditions; however, radiation stress, aging, sequential exposure to chemotherapy, and serial bone marrow transplantation increased senescence in animals lacking P2Y14. Enhanced senescence coincided with increased ROS, elevated p16(INK4a) expression, and hypophosphorylated Rb and was inhibited by treatment with a ROS scavenger or inhibition of p38/MAPK and JNK. Treatment of WT cells with pertussis toxin recapitulated the P2Y14 phenotype, suggesting that P2Y14 mediates antisenescence effects through Gi/o protein-dependent pathways. Primitive hematopoietic cells lacking P2Y14 were compromised in their ability to restore hematopoiesis in irradiated mice. Together, these data indicate that P2Y14 on stem/progenitor cells of the hematopoietic system inhibits cell senescence by monitoring and responding to the extracellular manifestations of tissue stress and suggest that P2Y14-mediated responses prevent the premature decline of regenerative capacity after injury.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/efectos de la radiación , Receptores Purinérgicos P2Y/metabolismo , Envejecimiento/metabolismo , Envejecimiento/patología , Envejecimiento/efectos de la radiación , Animales , Muerte Celular/fisiología , Muerte Celular/efectos de la radiación , Senescencia Celular/fisiología , Senescencia Celular/efectos de la radiación , Desarrollo Embrionario/fisiología , Desarrollo Embrionario/efectos de la radiación , Femenino , Hematopoyesis/fisiología , Hematopoyesis/efectos de la radiación , Células Madre Hematopoyéticas/citología , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Noqueados , Modelos Biológicos , Embarazo , Especies Reactivas de Oxígeno/metabolismo , Receptores Purinérgicos P2Y/deficiencia , Receptores Purinérgicos P2Y/genética , Transducción de Señal , Estrés Fisiológico/fisiología , Estrés Fisiológico/efectos de la radiaciónRESUMEN
Hematopoietic stem progenitor cells (HSPCs) are present in very small numbers in the circulating blood in steady-state conditions. In response to stress or injury, HSPCs are primed to migrate out of their niche to peripheral blood. Mobilized HSPCs are now commonly used as stem cell sources due to faster engraftment and reduced risk of posttransplant infection. In this study, we demonstrated that a nucleotide sugar, UDP-glucose, which is released into extracellular fluids in response to stress, mediates HSPC mobilization. UDP-glucose-mobilized cells possessed the capacity to achieve long-term repopulation in lethally irradiated animals and the ability to differentiate into multi-lineage blood cells. Compared with G-CSF-mobilized cells, UDP-glucose-mobilized cells preferentially supported long-term repopulation and exhibited lymphoid-biased differentiation, suggesting that UDP-glucose triggers the mobilization of functionally distinct subsets of HSPCs. Furthermore, co-administration of UDP-glucose and G-CSF led to greater HSPC mobilization than G-CSF alone. Administration of the antioxidant agent NAC significantly reduced UDP-glucose-induced mobilization, coinciding with a reduction in RANKL and osteoclastogenesis. These findings provide direct evidence demonstrating a potential role for UDP-glucose in HSPC mobilization and may provide an attractive strategy to improve the yield of stem cells in poor-mobilizing allogeneic or autologous donors.
Asunto(s)
Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/efectos de los fármacos , Uridina Difosfato Glucosa/farmacología , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Quimiotaxis , Factor Estimulante de Colonias de Granulocitos/farmacología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Osteoclastos/fisiología , Superóxidos/metabolismoRESUMEN
The longevity of organisms is maintained by stem cells. If an organism loses the ability to maintain a balance between quiescence and differentiation in the stem/progenitor cell compartment due to aging and/or stress, this may result in death or age-associated diseases, including cancer. Ewing sarcoma is the most lethal bone tumor in young patients and arises from primitive stem cells. Here, we demonstrated that endogenous Ewing sarcoma gene (Ews) is indispensable for stem cell quiescence, and that the ablation of Ews promotes the early onset of senescence in hematopoietic stem progenitor cells. The phenotypic and functional changes in Ews-deficient stem cells were accompanied by an increase in senescence-associated ß-galactosidase staining and a marked induction of p16(INK4a) compared with wild-type counterparts. With its relevance to cancer and possibly aging, EWS is likely to play a significant role in maintaining the functional capacity of stem cells and may provide further insight into the complexity of Ewing sarcoma in the context of stem cells.