RESUMO
The Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), also known as CD66a, is a member of the immunoglobulin superfamily. CEACAM1 was shown to be a prognostic marker in patients suffering from cancer. In this review, we summarize pre-clinical and clinical evidence linking CEACAM1 to tumorigenicity and cancer progression. Furthermore, we discuss potential CEACAM1-based mechanisms that may affect cancer biology.
Assuntos
Moléculas de Adesão Celular , Neoplasias , Humanos , Antígenos CD/metabolismo , Antígeno Carcinoembrionário/metabolismo , Proteína CEACAM1 , Moléculas de Adesão Celular/metabolismoRESUMO
Structural maintenance of chromosomes flexible hinge domain-containing protein 1 (SMCHD1) has been implicated in X-chromosome inactivation, imprinting, and DNA damage repair, and mutations in SMCHD1 can cause facioscapulohumeral muscular dystrophy. More recently, SMCHD1 has also been identified as a component of telomeric chromatin. Here, we report that SMCHD1 is required for DNA damage signaling and non-homologous end joining (NHEJ) at unprotected telomeres. Co-depletion of SMCHD1 and the shelterin subunit TRF2 reduced telomeric 3'-overhang removal in time-course experiments, as well as the number of chromosome end fusions. SMCHD1-deficient cells displayed reduced ATM S1981 phosphorylation and diminished formation of γH2AX foci and of 53BP1-containing telomere dysfunction-induced foci (TIFs), indicating defects in DNA damage checkpoint signaling. Removal of TPP1 and subsequent activation of ATR signaling rescued telomere fusion events in TRF2-depleted SMCHD1 knockout cells. Together, these data indicate that SMCHD1 depletion reduces telomere fusions in TRF2-depleted cells due to defects in ATM-dependent checkpoint signaling and that SMCHD1 mediates DNA damage response activation upstream of ATM phosphorylation at uncapped telomeres.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/genética , Dano ao DNA , Reparo do DNA por Junção de Extremidades , Epistasia Genética , Técnicas de Inativação de Genes , Células HeLa , Humanos , Fosforilação , Complexo Shelterina/genética , Complexo Shelterina/metabolismo , Transdução de Sinais , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
Aging is an independent risk factor for cardiovascular diseases and therefore of particular interest for the prevention of cardiovascular events. However, the mechanisms underlying vascular aging are not well understood. Since carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is crucially involved in vascular homeostasis, we sought to identify the role of CEACAM1 in vascular aging. Using human internal thoracic artery and murine aorta, we show that CEACAM1 is upregulated in the course of vascular aging. Further analyses demonstrated that TNF-α is CEACAM1-dependently upregulated in the aging vasculature. Vice versa, TNF-α induces CEACAM1 expression. This results in a feed-forward loop in the aging vasculature that maintains a chronic pro-inflammatory milieu. Furthermore, we demonstrate that age-associated vascular alterations, that is, increased oxidative stress and vascular fibrosis, due to increased medial collagen deposition crucially depend on the presence of CEACAM1. Additionally, age-dependent upregulation of vascular CEACAM1 expression contributes to endothelial barrier impairment, putatively via increased VEGF/VEGFR-2 signaling. Consequently, aging-related upregulation of vascular CEACAM1 expression results in endothelial dysfunction that may promote atherosclerotic plaque formation in the presence of additional risk factors. Our data suggest that CEACAM1 might represent an attractive target in order to delay physiological aging and therefore the transition to vascular disorders such as atherosclerosis.
Assuntos
Envelhecimento/metabolismo , Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Endotélio Vascular/metabolismo , Transdução de Sinais , Idoso , Animais , Antígenos CD/genética , Moléculas de Adesão Celular/genética , Células Cultivadas , Endotélio Vascular/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Incidence of childhood cancer increased in most countries worldwide, but reasons are unclear. This study investigates trends of childhood cancer incidence in Switzerland from 1985 to 2014. METHODS: We extracted data on all childhood cancer cases diagnosed at ages 0-14 years in Switzerland from the Swiss Childhood Cancer Registry. We included ICCC-3 main groups I-XII and calculated age-standardised, cumulative, and age-specific incidence for different diagnostic groups. We analysed trends of annual age-standardised incidence using JoinPoint regression models. RESULTS: Over the study period from 1985 to 2014, 5104 of 5486 cancer diagnoses (93%) were microscopically verified. The proportion of children treated in paediatric cancer centres increased from 84% during 1985-1994 to 93% in 1995-2004 and 98% in 2005-2014 (pâ¯<â¯0.001). Using the World standard population, age-standardised incidence was 143 in 1985-1994, 154 in 1995-2004, and 162 per million in 2005-2014. Incidence increased by 0.7% (95% confidence interval (CI) 0.5; 1.0) per year for all cancers from 1985 to 2014, 0.8% (95% CI 0.2%-1.4%) for leukaemias over the same period, 3.0% (95% CI 0.2%-1.4%) for CNS tumours during 1985-2002, and 3.8% (95% CI 1.7%-6.0%) for epithelial neoplasms and melanomas over the period 1985-2014. CONCLUSION: Trends in incidence were driven mostly by increases among leukaemias and CNS tumours. For CNS tumours, observed trends may be explained at least partially by diagnostic changes and improved registration. For leukaemias, rising incidence may be real and due to risk factors that experience similar increases in trends.
Assuntos
Sobreviventes de Câncer/estatística & dados numéricos , Adolescente , Criança , Pré-Escolar , Feminino , História do Século XX , História do Século XXI , Humanos , Incidência , Lactente , Recém-Nascido , Masculino , Fatores de Risco , SuíçaRESUMO
RATIONALE: Regeneration of lost cardiomyocytes is a fundamental unresolved problem leading to heart failure. Despite several strategies developed from intensive studies performed in the past decades, endogenous regeneration of heart tissue is still limited and presents a big challenge that needs to be overcome to serve as a successful therapeutic option for myocardial infarction. OBJECTIVE: One of the essential prerequisites for cardiac regeneration is the identification of endogenous cardiomyocyte progenitors and their niche that can be targeted by new therapeutic approaches. In this context, we hypothesized that the vascular wall, which was shown to harbor different types of stem and progenitor cells, might serve as a source for cardiac progenitors. METHODS AND RESULTS: We describe generation of spontaneously beating mouse aortic wall-derived cardiomyocytes without any genetic manipulation. Using aortic wall-derived cells (AoCs) of WT (wild type), αMHC (α-myosin heavy chain), and Flk1 (fetal liver kinase 1)-reporter mice and magnetic bead-associated cell sorting sorting of Flk1+ AoCs from GFP (green fluorescent protein) mice, we identified Flk1+CD (cluster of differentiation) 34+Sca-1 (stem cell antigen-1)-CD44- AoCs as the population that gives rise to aortic wall-derived cardiomyocytes. This AoC subpopulation delivered also endothelial cells and macrophages with a particular accumulation within the aortic wall-derived cardiomyocyte containing colonies. In vivo, cardiomyocyte differentiation capacity was studied by implantation of fluorescently labeled AoCs into chick embryonic heart. These cells acquired cardiomyocyte-like phenotype as shown by αSRA (α-sarcomeric actinin) expression. Furthermore, coronary adventitial Flk1+ and CD34+ cells proliferated, migrated into the myocardium after mouse myocardial infarction, and expressed Isl-1+ (insulin gene enhancer protein-1) indicative of cardiovascular progenitor potential. CONCLUSIONS: Our data suggest Flk1+CD34+ vascular adventitia-resident stem cells, including those of coronary adventitia, as a novel endogenous source for generating cardiomyocytes. This process is essentially supported by endothelial cells and macrophages. In summary, the therapeutic manipulation of coronary adventitia-resident cardiac stem and their supportive cells may open new avenues for promoting cardiac regeneration and repair after myocardial infarction and for preventing heart failure.
Assuntos
Túnica Adventícia/citologia , Aorta Torácica/citologia , Diferenciação Celular , Proliferação de Células , Miócitos Cardíacos/fisiologia , Células-Tronco/fisiologia , Animais , Antígenos CD34/metabolismo , Antígenos Ly/metabolismo , Células Cultivadas , Embrião de Galinha , Modelos Animais de Doenças , Feminino , Genes Reporter , Separação Imunomagnética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/cirurgia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/transplante , Cadeias Pesadas de Miosina/genética , Fenótipo , Regeneração , Transplante de Células-Tronco , Células-Tronco/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Miosinas Ventriculares/genéticaRESUMO
Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is known to be crucial to vasculogenesis and angiogenesis. Recently, CEACAM1 deficiency was shown to result in the formation of aortic plaque-like lesions, indicating a role for CEACAM1 in adult vessels as well. The underlying mechanisms remained largely elusive. Therefore, we aimed to elucidate the role of CEACAM1 in endothelial homeostasis. Here, we show that CEACAM1 deficiency causes subcellular eNOS redistribution in endothelial cells ( i.e., by eNOS depalmitoylation) and alters endothelial glycocalyx that confers antiadhesive properties to the endothelium ( i.e., by repression of glycocalyx-degrading enzymes). Accordingly, our analysis revealed an increased leukocyte-endothelial interaction in CEACAM1-deficient endothelium. In addition, CEACAM1 age dependently modulated basal and TNF-α-mediated endothelial barrier (EB) leakiness. In younger mice, CEACAM1 was protective for EB, whereas in aged mice it promoted EB leakiness. EB function depends on interendothelial adherence junctions formed by ß-catenin/vascular endothelial-cadherin complexes. We show here that CEACAM1 influenced basal and TNF-α-mediated phosphorylation of ß-catenin and caveolin-1, which are essential players in EB modulation. Both increased adhesiveness to leukocytes and EB modulation due to CEACAM1 deficiency may facilitate inflammatory cell transmigration into the vascular wall and subsequent plaque formation. Collectively, these results identify a crucial role for CEACAM1 in endothelial homeostasis of adult blood vessels.-Ghavampour, S., Kleefeldt, F., Bömmel, H., Volland, J., Paus, A., Horst, A., Pfeiffer, V., Hübner, S., Wagner, N., Rueckschloss, U., Ergün, S. Endothelial barrier function is differentially regulated by CEACAM1-mediated signaling.
Assuntos
Antígeno Carcinoembrionário/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Transdução de Sinais , Migração Transendotelial e Transepitelial , Animais , Caderinas/metabolismo , Caveolina 1/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/patologia , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
RAF kinases are major constituents of the mitogen activated signaling pathway, regulating cell proliferation, differentiation and cell survival of many cell types, including neurons. In mammals, the family of RAF proteins consists of three members, ARAF, BRAF, and CRAF. Ablation of CRAF kinase in inbred mouse strains causes major developmental defects during fetal growth and embryonic or perinatal lethality. Heterozygous germline mutations in CRAF result in Noonan syndrome, which is characterized by neurocognitive impairment that may involve hippocampal physiology. The role of CRAF signaling during hippocampal development and generation of new postnatal hippocampal granule neurons has not been examined and may provide novel insight into the cause of hippocampal dysfunction in Noonan syndrome. In this study, by crossing CRAF-deficiency to CD-1 outbred mice, a CRAF mouse model was established which enabled us to investigate the interplay of neural progenitor proliferation and postmitotic differentiation during adult neurogenesis in the hippocampus. Albeit the general morphology of the hippocampus was unchanged, CRAF-deficient mice displayed smaller granule cell layer (GCL) volume at postnatal day 30 (P30). In CRAF-deficient mice a substantial number of abnormal, chromophilic, fast dividing cells were found in the subgranular zone (SGZ) and hilus of the dentate gyrus (DG), indicating that CRAF signaling contributes to hippocampal neural progenitor proliferation. CRAF-deficient neural progenitor cells showed an increased cell death rate and reduced neuronal maturation. These results indicate that CRAF function affects postmitotic neural cell differentiation and points to a critical role of CRAF-dependent growth factor signaling pathway in the postmitotic development of adult-born neurons.
Assuntos
Diferenciação Celular , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Animais , Animais Recém-Nascidos , Proliferação de Células/genética , Giro Denteado/citologia , Giro Denteado/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/genética , Neurogênese/genética , Neurônios/citologia , Proteínas Proto-Oncogênicas c-raf/genética , Fatores de TempoRESUMO
Telomeres are specialized nucleoprotein structures that protect chromosome ends from DNA damage response (DDR) and DNA rearrangements. The telomeric shelterin protein TRF2 suppresses the DDR, and this function has been attributed to its abilities to trigger t-loop formation or prevent massive decompaction and loss of density of telomeric chromatin. Here, we applied stochastic optical reconstruction microscopy (STORM) to measure the sizes and shapes of functional human telomeres of different lengths and dysfunctional telomeres that elicit a DDR. Telomeres have an ovoid appearance with considerable plasticity in shape. Examination of many telomeres demonstrated that depletion of TRF2, TRF1, or both affected the sizes of only a small subset of telomeres. Costaining of telomeres with DDR markers further revealed that the majority of DDR signaling telomeres retained a normal size. Thus, DDR signaling at telomeres does not require decompaction. We propose that telomeres are monitored by the DDR machinery in the absence of telomere expansion and that the DDR is triggered by changes at the molecular level in structure and protein composition.
Assuntos
Dano ao DNA , Telômero/ultraestrutura , Cromatina/fisiologia , Imunofluorescência , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Microscopia de Fluorescência , Proteína 1 de Ligação a Repetições Teloméricas/análise , Proteína 1 de Ligação a Repetições Teloméricas/imunologia , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/fisiologiaRESUMO
Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes hepatic insulin clearance. Consistently, mice with null mutation of Ceacam1 (Cc1(-/-)) exhibit impaired insulin clearance with increased lipid production in liver and redistribution to white adipose tissue, leading to visceral obesity at 2 months of age. When the mutation is propagated on the C57/BL6J genetic background, total fat mass rises significantly with age, and glucose intolerance and systemic insulin resistance develop at 6 months of age. This study was carried out to determine the mechanisms underlying the marked increase in total fat mass in 6-month-old mutants. Indirect calorimetry analysis showed that Cc1(-/-) mice develop hyperphagia and a significant reduction in physical activity, in particular in the early hours of the dark cycle, during which energy expenditure is only slightly lower than in wild-type mice. They also exhibit increased triglyceride accumulation in skeletal muscle, due in part to incomplete fatty acid ß-oxidation. Mechanistically, hypothalamic leptin signaling is reduced, as demonstrated by blunted STAT3 phosphorylation in coronal sections in response to an intracerebral ventricular injection of leptin. Hypothalamic fatty-acid synthase activity is also elevated in the mutants. Together, the data show that the increase in total fat mass in Cc1(-/-) mice is mainly attributed to hyperphagia and reduced spontaneous physical activity. Although the contribution of the loss of CEACAM1 from anorexigenic proopiomelanocortin neurons in the arcuate nucleus is unclear, leptin resistance and elevated hypothalamic fatty-acid synthase activity could underlie altered energy balance in these mice.
Assuntos
Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/metabolismo , Leptina/metabolismo , Obesidade/etiologia , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Moléculas de Adesão Celular/deficiência , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Metabolismo Energético , Ácidos Graxos/metabolismo , Deleção de Genes , Hiperfagia/etiologia , Hiperfagia/genética , Hiperfagia/metabolismo , Hipotálamo/metabolismo , Resistência à Insulina , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/metabolismo , Mutação , Obesidade/genética , Obesidade/metabolismo , Pró-Opiomelanocortina/metabolismo , Transdução de Sinais , Triglicerídeos/metabolismoRESUMO
Carcinoembryonic antigen-related cell adhesion molecule 2 (CEACAM2) regulates food intake as demonstrated by hyperphagia in mice with the Ceacam2 null mutation (Cc2(-/-)). This study investigated whether CEACAM2 also regulates insulin secretion. Ceacam2 deletion caused an increase in ß-cell secretory function, as assessed by hyperglycemic clamp analysis, without affecting insulin response. Although CEACAM2 is expressed in pancreatic islets predominantly in non-ß-cells, basal plasma levels of insulin, glucagon and somatostatin, islet areas, and glucose-induced insulin secretion in pooled Cc2(-/-) islets were all normal. Consistent with immunofluorescence analysis showing CEACAM2 expression in distal intestinal villi, Cc2(-/-) mice exhibited a higher release of oral glucose-mediated GLP-1, an incretin that potentiates insulin secretion in response to glucose. Compared with wild type, Cc2(-/-) mice also showed a higher insulin excursion during the oral glucose tolerance test. Pretreating with exendin(9-39), a GLP-1 receptor antagonist, suppressed the effect of Ceacam2 deletion on glucose-induced insulin secretion. Moreover, GLP-1 release into the medium of GLUTag enteroendocrine cells was increased with siRNA-mediated Ceacam2 down-regulation in parallel to an increase in Ca(2+) entry through L-type voltage-dependent Ca(2+) channels. Thus, CEACAM2 regulates insulin secretion, at least in part, by a GLP-1-mediated mechanism, independent of confounding metabolic factors.
Assuntos
Moléculas de Adesão Celular/deficiência , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Glucose/farmacologia , Animais , Antígenos CD/metabolismo , Canais de Cálcio Tipo L/metabolismo , Moléculas de Adesão Celular/metabolismo , Imunofluorescência , Teste de Tolerância a Glucose , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , VigíliaRESUMO
The family of RAF kinases transduces extracellular information to the nucleus, and their activation is crucial for cellular regulation on many levels, ranging from embryonic development to carcinogenesis. B-RAF and C-RAF modulate neurogenesis and neuritogenesis during chicken inner ear development. C-RAF deficiency in humans is associated with deafness in the rare genetic insulin-like growth factor 1 (IGF-1), Noonan and Leopard syndromes. In this study, we show that RAF kinases are expressed in the developing inner ear and in adult mouse cochlea. A homozygous C-Raf deletion in mice caused profound deafness with no evident cellular aberrations except for a remarkable reduction of the K(+) channel Kir4.1 expression, a trait that suffices as a cause of deafness. To explore the role of C-Raf in cellular protection and repair, heterozygous C-Raf (+/-) mice were exposed to noise. A reduced C-RAF level negatively affected hearing preservation in response to noise through mechanisms involving the activation of JNK and an exacerbated apoptotic response. Taken together, these results strongly support a role for C-RAF in hearing protection.
Assuntos
Orelha Interna/metabolismo , Perda Auditiva/genética , Ruído , Proteínas Proto-Oncogênicas c-raf/fisiologia , Animais , Apoptose/genética , Cóclea/metabolismo , Orelha Interna/embriologia , Feminino , Perda Auditiva/metabolismo , Masculino , Camundongos , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Transdução de SinaisRESUMO
Tumors are often greatly dependent on signaling cascades promoting cell growth or survival and may become hypersensitive to inactivation of key components within these signaling pathways. Ras and RAF mutations found in human cancer confer constitutive activity to these signaling molecules thereby converting them into an oncogenic state. RAF dimerization is required for normal Ras-dependent RAF activation and is required for the oncogenic potential of mutant RAFs. Here we describe a new mouse model for lung tumor development to investigate the role of B-RAF in oncogenic C-RAF-mediated adenoma initiation and growth. Conditional elimination of B-RAF in C-RAF BxB-expressing embryonic alveolar epithelial type II cells did not block adenoma formation. However, loss of B-RAF led to significantly reduced tumor growth. The diminished tumor growth upon B-RAF inactivation was due to reduced cell proliferation in absence of senescence and increased apoptosis. Furthermore, B-RAF elimination inhibited C-RAF BxB-mediated activation of the mitogenic cascade. In line with these data, mutation of Ser-621 in C-RAF BxB abrogated in vitro the dimerization with B-RAF and blocked the ability to activate the MAPK cascade. Taken together these data indicate that B-RAF is an important factor in oncogenic C-RAF-mediated tumorigenesis.
Assuntos
Adenoma/enzimologia , Transformação Celular Neoplásica/metabolismo , Células Epiteliais/enzimologia , Neoplasias Pulmonares/enzimologia , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Alvéolos Pulmonares/enzimologia , Mucosa Respiratória/enzimologia , Adenoma/genética , Adenoma/patologia , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Epiteliais/patologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Transgênicos , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/genética , Alvéolos Pulmonares/patologia , Mucosa Respiratória/patologiaRESUMO
Telomere maintenance by the conventional DNA replication machinery and telomerase is assisted by specialized DNA helicases, nucleases and telomere binding proteins. Here, we identify the THO components at telomeres and define critical roles of this complex in telomere stability. Deletion of the THO-subunit THP2 leads to telomere shortening. We discover that telomeres contain RNA:DNA hybrid structures or R-loops which involve the long-noncoding RNA TERRA and which accumulate in thp2-Δ cells. Telomere length is not restored by R-loop removal upon RNase H overexpression, but by deletion of Exonuclease 1 (Exo1). Replication stress further enhances the short telomere phenotype of THP2 mutants. Similar events occur upon induced transcription of TERRA and genetic analysis links Thp2 to TERRA function. Altogether, our data indicate that THO, through the interplay with TERRA, regulates chromosome end processing activities and prevents interference with semiconservative DNA replication of telomeric DNA.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Telômero/fisiologia , DNA Fúngico/genética , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Telômero/química , Encurtamento do Telômero/genética , Fatores de Transcrição/metabolismoRESUMO
Telomeres are the physical ends of eukaryotic chromosomes. They protect chromosome ends from DNA degradation, recombination, and DNA end fusions, and they are important for nuclear architecture. Telomeres provide a mechanism for their replication by semiconservative DNA replication and length maintenance by telomerase. Through telomerase repression and induced telomere shortening, telomeres provide the means to regulate cellular life span. In this review, we introduce the current knowledge on telomere composition and structure. We then discuss in depth the current understanding of how telomere components mediate their function during semiconservative DNA replication and how telomerase is regulated at the end of the chromosome. We focus our discussion on the telomeres from mammals and the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe.
Assuntos
Replicação do DNA/fisiologia , Telomerase/metabolismo , Homeostase do Telômero , Telômero/fisiologia , Animais , Mamíferos/genética , Modelos Genéticos , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Telômero/químicaRESUMO
This study focuses on the role of the kinase BRaf in postnatal brain development. Mice expressing truncated, non-functional BRaf in neural stem cell-derived brain tissue demonstrate alterations in the cerebellum, with decreased sizes and fuzzy borders of the glomeruli in the granule cell layer. In addition we observed reduced numbers and misplaced ectopic Purkinje cells that showed an altered structure of their dendritic arborizations in the hippocampus, while the overall cornus ammonis architecture appeared to be unchanged. In male mice lacking BRaf in the hippocampus the size of the granule cell layer was normal at postnatal day 12 (P12) but diminished at P21, as compared to control littermates. This defect was caused by a reduced ability of dentate gyrus progenitor cells to differentiate into NeuN positive granule cell neurons. In vitro cell culture of P0/P1 hippocampal cells revealed that BRaf deficient cells were impaired in their ability to form microtubule-associated protein 2 positive neurons. Together with the alterations in behaviour, such as autoaggression and loss of balance fitness, these observations indicate that in the absence of BRaf all neuronal cellular structures develop, but neuronal circuits in the cerebellum and hippocampus are partially disturbed besides impaired neuronal generation in both structures.
Assuntos
Diferenciação Celular/genética , Cerebelo/metabolismo , Hipocampo/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Animais Recém-Nascidos , Comportamento Animal , Cerebelo/crescimento & desenvolvimento , Giro Denteado/crescimento & desenvolvimento , Giro Denteado/metabolismo , Deleção de Genes , Hipocampo/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Transgênicos , FenótipoRESUMO
Numerous small non-coding RNAs (sRNAs) in bacteria modulate rates of translation initiation and degradation of target mRNAs, which they recognize through base-pairing facilitated by the RNA chaperone Hfq. Recent evidence indicates that the ternary complex of Hfq, sRNA and mRNA guides endoribonuclease RNase E to initiate turnover of both the RNAs. We show that a sRNA not only guides RNase E to a defined site in a target RNA, but also allosterically activates the enzyme by presenting a monophosphate group at the 5'-end of the cognate-pairing "seed." Moreover, in the absence of the target the 5'-monophosphate makes the sRNA seed region vulnerable to an attack by RNase E against which Hfq confers no protection. These results suggest that the chemical signature and pairing status of the sRNA seed region may help to both 'proofread' recognition and activate mRNA cleavage, as part of a dynamic process involving cooperation of RNA, Hfq and RNase E.
Assuntos
Endorribonucleases/metabolismo , Porinas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Endorribonucleases/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Pequeno RNA não Traduzido/metabolismo , Salmonella/genética , Salmonella/metabolismoRESUMO
The long noncoding telomeric repeat containing RNA (TERRA) is expressed at chromosome ends. TERRA upregulation upon experimental manipulation or in ICF (immunodeficiency, centromeric instability, facial anomalies) patients correlates with short telomeres. To study the mechanism of telomere length control by TERRA in Saccharomyces cerevisiae, we mapped the transcriptional start site of TERRA at telomere 1L and inserted a doxycycline regulatable promoter upstream. Induction of TERRA transcription led to telomere shortening of 1L but not of other chromosome ends. TERRA interacts with the Exo1-inhibiting Ku70/80 complex, and deletion of EXO1 but not MRE11 fully suppressed the TERRA-mediated short telomere phenotype in presence and absence of telomerase. Thus TERRA transcription facilitates the 5'-3' nuclease activity of Exo1 at chromosome ends, providing a means to regulate the telomere shortening rate. Thereby, telomere transcription can regulate cellular lifespan through modulation of chromosome end processing activities.
Assuntos
Cromossomos/genética , Saccharomyces cerevisiae/genética , Encurtamento do Telômero , Telômero/genética , Transcrição Gênica , Aldose-Cetose Isomerases/genética , Sequência de Bases , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Proteínas de Saccharomyces cerevisiae/genética , Telomerase/genética , Telomerase/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
Telomeric repeat-containing RNA (TERRA) has been implicated in the control of heterochromatin and telomerase. We demonstrate that yeast TERRA is regulated by telomere-binding proteins in a chromosome-end-specific manner that is dependent on subtelomeric repetitive DNA elements. At telomeres that contain only X-elements, the Rap1 carboxy-terminal domain recruits the Sir2/3/4 and Rif1/2 complexes to repress transcription in addition to promoting Rat1-nuclease-dependent TERRA degradation. At telomeres that contain Y' elements, however, Rap1 represses TERRA through recruitment of Rif1 and Rif2. Our work emphasizes the importance of subtelomeric DNA in the control of telomeric protein composition and telomere transcription.
Assuntos
Regulação Fúngica da Expressão Gênica , Sequências Repetitivas de Ácido Nucleico/genética , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Fatores de Transcrição/metabolismo , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , Mutação/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Complexo Shelterina , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genéticaRESUMO
Bacterial small noncoding RNAs (sRNAs) generally recognize target mRNAs in the 5' region to prevent 30S ribosomes from initiating translation. It was thought that the mRNA coding sequence (CDS) was refractory to sRNA-mediated repression, because elongating 70S ribosomes have an efficient RNA helicase activity that prevents stable target pairing. We report that the Hfq-associated MicC sRNA silences Salmonella typhimurium ompD mRNA via a Assuntos
Fases de Leitura Aberta/genética
, Biossíntese de Proteínas/genética
, RNA Mensageiro/genética
, RNA não Traduzido/genética
, Proteínas de Bactérias/genética
, Proteínas de Bactérias/metabolismo
, Sequência de Bases
, Northern Blotting
, Western Blotting
, Eletroforese em Gel de Poliacrilamida
, Endorribonucleases/metabolismo
, Perfilação da Expressão Gênica
, Regulação Bacteriana da Expressão Gênica
, Dados de Sequência Molecular
, Mutação
, Iniciação Traducional da Cadeia Peptídica/genética
, Porinas/genética
, Estabilidade de RNA
, RNA Bacteriano/genética
, RNA Bacteriano/metabolismo
, RNA Mensageiro/metabolismo
, RNA não Traduzido/metabolismo
, Reação em Cadeia da Polimerase Via Transcriptase Reversa
, Ribossomos/metabolismo
, Salmonella typhimurium/genética
, Salmonella typhimurium/metabolismo
RESUMO
Post-transcriptional repression of porin synthesis has emerged as a major function of Hfq-dependent, small non-coding RNAs (sRNAs). Many enterobacteria express OmpX-like porins, a family of outer membrane proteins whose physiological roles and structural properties have been studied intensively. While regulatory sRNAs have been identified for most major and many minor porins of Salmonella and Escherichia coli, a post-transcriptional regulator of OmpX levels has never been found. Here, we have taken a 'reverse target search' approach by systematic inactivation of Salmonella sRNA genes, and screening 35 sRNA deletion strains for effects on OmpX synthesis. We have identified the Hfq-dependent CyaR (formerly RyeE) sRNA as an ompX repressor. Global transcriptomic profiling following induction of CyaR expression suggests that ompX mRNA is the primary target of this sRNA under standard growth conditions. The results of phylogenetic and mutational analyses suggest that a conserved RNA hairpin of CyaR, featuring a C-rich apical loop, acts to sequester the Shine-Dalgarno sequence of ompX mRNA and to inhibit translational initiation. We have also discovered that cyaR expression is tightly controlled by the cyclic AMP receptor protein, CRP. This represents a new link between porin repression and nutrient availability that is likely to be widely conserved among enterobacteria.