RESUMO
The role of telomerase reverse transcriptase (TERT) induction and telomere maintenance in carcinogenesis including cervical cancer (CC) pathogenesis has been well established. However, it remains unclear whether they affect infection of high-risk human papillomavirus (hrHPV), an initiating event for CC development. Similarly, genetic variants at the TERT locus are shown to be associated with susceptibility to CC, but it is unclear whether these SNPs modify the risk for cervical HPV infection. Here we show that in CC-derived HeLa cells, TERT overexpression inhibits, while its depletion upregulates expression of Syndecan-1 (SDC-1), a key component for HPV entry receptors. The TCGA cohort of CC analyses reveals an inverse correlation between TERT and SDC-1 expression (R = -0.23, P = 0.001). We further recruited 1330 females (520 non-HPV and 810 hrHPV-infected) without CC or high-grade cervical intraepithelial neoplasia to analyze telomeres in cervical epithelial cells and SNPs at rs2736098, rs2736100 and rs2736108, previously identified TERT SNPs for CC risk. Non-infected females exhibited age-related telomere shortening in cervical epithelial cells and their telomeres were significantly longer than those in hrHPV-infected group (1.31 ± 0.62 vs 1.19 ± 0.48, P < 0.001). There were no differences in rs2736098 and rs2736100 genotypes, but non-infected individuals had significantly a higher C-allele frequency (associated with higher TERT expression) while lower T-allele levels at rs2736108 compared with those in the hrHPV group (P = 0.020). Collectively, appropriate telomere maintenance and TERT expression in normal cervical cells may prevent CC by modulating hrHPV infection predisposition, although they are required for CC development and progression.
Assuntos
Predisposição Genética para Doença/genética , Infecções por Papillomavirus/genética , Telomerase/genética , Telômero/genética , Neoplasias do Colo do Útero/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Epitélio/metabolismo , Epitélio/virologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologia , Polimorfismo de Nucleotídeo Único , Telomerase/metabolismo , Telômero/enzimologia , Neoplasias do Colo do Útero/complicações , Neoplasias do Colo do Útero/metabolismo , Adulto JovemRESUMO
Telomeres are part of a highly refined system for maintaining the stability of linear chromosomes. Most telomeres rely on simple repetitive sequences and telomerase enzymes to protect chromosomal ends; however, in some species or telomerase-defective situations, an alternative lengthening of telomeres (ALT) mechanism is used. ALT mainly utilises recombination-based replication mechanisms and the constituents of ALT-based telomeres vary depending on models. Here we show that mouse telomeres can exploit non-telomeric, unique sequences in addition to telomeric repeats. We establish that a specific subtelomeric element, the mouse template for ALT (mTALT), is used for repairing telomeric DNA damage as well as for composing portions of telomeres in ALT-dependent mouse embryonic stem cells. Epigenomic and proteomic analyses before and after ALT activation reveal a high level of non-coding mTALT transcripts despite the heterochromatic nature of mTALT-based telomeres. After ALT activation, the increased HMGN1, a non-histone chromosomal protein, contributes to the maintenance of telomere stability by regulating telomeric transcription. These findings provide a molecular basis to study the evolution of new structures in telomeres.
Assuntos
Células-Tronco Embrionárias Murinas/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Telomerase/genética , Homeostase do Telômero/genética , Telômero/genética , Animais , Proteínas de Ligação a DNA/genética , Epigenômica/métodos , Células HEK293 , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Proteômica/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Telomerase/metabolismo , Telômero/enzimologia , Fatores de Transcrição/genéticaRESUMO
Telomerase is an attractive target for anti-tumor therapy as it is almost universally expressed in cancer cells. Here, we show that treatment with a telomere-targeting drug, 6-thio-2'-deoxyguanosine (6-thio-dG), leads to tumor regression through innate and adaptive immune-dependent responses in syngeneic and humanized mouse models of telomerase-expressing cancers. 6-thio-dG treatment causes telomere-associated DNA damages that are sensed by dendritic cells (DCs) and activates the host cytosolic DNA sensing STING/interferon I pathway, resulting in enhanced cross-priming capacity of DCs and tumor-specific CD8+ T cell activation. Moreover, 6-thio-dG overcomes resistance to checkpoint blockade in advanced cancer models. Our results unveil how telomere stress increases innate sensing and adaptive anti-tumor immunity and provide strong rationales for combining telomere-targeting therapy with immunotherapy.
Assuntos
Desoxiguanosina/análogos & derivados , Proteínas de Membrana/imunologia , Neoplasias/tratamento farmacológico , Telomerase/antagonistas & inibidores , Telômero/genética , Tionucleosídeos/farmacologia , Imunidade Adaptativa/efeitos dos fármacos , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Desoxiguanosina/farmacologia , Desoxiguanosina/uso terapêutico , Células HCT116 , Humanos , Imunidade Inata/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/genética , Neoplasias/imunologia , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Telomerase/metabolismo , Telômero/enzimologia , Tionucleosídeos/uso terapêutico , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Carga Tumoral/imunologiaRESUMO
BACKGROUND: Human telomerase reverse transcriptase (hTERT) and its components play a significant role in cancer progression, but recent data demonstrated that telomeres and telomerase alterations could be found in other diseases; increasing evidence suggests a key role of this enzyme in the fields of hepatobiliary and pancreatic diseases. DATA SOURCES: We performed a PubMed search with the following keywords: telomerase, hepatocellular carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma by December 2019. We reviewed the relevant publications that analyzed the correlation between telomerase activity and hepatobiliary and pancreatic diseases. RESULTS: Telomerase reactivation plays a significant role in the development and progression of hepatobiliary and pancreatic tumors and could be used as a diagnostic biomarker for hepatobiliary and pancreatic cancers, as a predictor for prognosis and a promising therapeutic target. CONCLUSIONS: Our review summarized the evidence about the critical role of hTERT in cancerous and precancerous lesions of the alteration and its activity in hepatobiliary and pancreatic diseases.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias do Sistema Digestório/enzimologia , Telomerase/metabolismo , Homeostase do Telômero , Telômero/enzimologia , Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Animais , Neoplasias dos Ductos Biliares/enzimologia , Neoplasias dos Ductos Biliares/genética , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Colangiocarcinoma/enzimologia , Colangiocarcinoma/genética , Neoplasias do Sistema Digestório/genética , Ativação Enzimática , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Prognóstico , Telomerase/genética , Telômero/metabolismoRESUMO
T cells play a critical role in controlling viral infection; however, the mechanisms regulating their responses remain incompletely understood. Here, we investigated the role of topoisomerase IIA (Top2α, an enzyme that is essential in resolving entangled DNA strands during replication) in telomeric DNA damage and T cell dysfunction during viral infection. We demonstrated that T cells derived from patients with chronic viral (HBV, HCV, and HIV) infection had lower Top2α protein levels and enzymatic activity, along with an accumulation of the Top2α cleavage complex (Top2cc) in genomic DNA. In addition, T cells from virally infected subjects with lower Top2α levels were vulnerable to Top2α inhibitor-induced cell apoptosis, indicating an important role for Top2α in preventing DNA topological disruption and cell death. Using Top2α inhibitor (ICRF193 or Etoposide)-treated primary T cells as a model, we demonstrated that disrupting the DNA topology promoted DNA damage and T cell apoptosis via Top2cc accumulation that is associated with protein-DNA breaks (PDB) at genomic DNA. Disruption of the DNA topology was likely due to diminished expression of tyrosyl-DNA phosphodiesterase 2 (TDP2), which was inhibited in T cells in vitro by Top2α inhibitor and in vivo by chronic viral infection. These results suggest that immune-evasive viruses (HBV, HCV, and HIV) can disrupt T cell DNA topology as a mechanism of dysregulating host immunity and establishing chronic infection. Thus, restoring the DNA topologic machinery may serve as a novel strategy to protect T cells from unwanted DNA damage and to maintain immune competence.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Dano ao DNA , DNA Topoisomerases Tipo II/metabolismo , Viroses/genética , Viroses/imunologia , Adulto , Idoso , Apoptose/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/enzimologia , Doença Crônica , Reparo do DNA , DNA Topoisomerases Tipo II/biossíntese , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Dicetopiperazinas , Etoposídeo/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Diester Fosfórico Hidrolases/metabolismo , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Telomerase/antagonistas & inibidores , Telomerase/metabolismo , Telômero/efeitos dos fármacos , Telômero/enzimologia , Telômero/genética , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Inibidores da Topoisomerase II/farmacologia , Viroses/enzimologia , Adulto JovemRESUMO
Telomere maintenance by telomerase is essential for continuous proliferation of human cells and is vital for the survival of stem cells and 90% of cancer cells. To compensate for telomeric DNA lost during DNA replication, telomerase processively adds GGTTAG repeats to chromosome ends by copying the template region within its RNA subunit. Between repeat additions, the RNA template must be recycled. How telomerase remains associated with substrate DNA during this critical translocation step remains unknown. Using a single-molecule telomerase activity assay utilizing high-resolution optical tweezers, we demonstrate that stable substrate DNA binding at an anchor site within telomerase facilitates the processive synthesis of telomeric repeats. The product DNA synthesized by telomerase can be recaptured by the anchor site or fold into G-quadruplex structures. Our results provide detailed mechanistic insights into telomerase catalysis, a process of critical importance in aging and cancer.
Assuntos
DNA/metabolismo , Quadruplex G , RNA/metabolismo , Telomerase/metabolismo , Telômero/enzimologia , Biocatálise , DNA/genética , Replicação do DNA , Expressão Gênica , Células HEK293 , Humanos , Pinças Ópticas , RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Telomerase/genética , Telômero/ultraestruturaRESUMO
In both mammalian and fission yeast cells, conserved shelterin and CST (CTC1-STN1-TEN1) complexes play critical roles in protection of telomeres and regulation of telomerase, an enzyme required to overcome the end replication problem. However, molecular details that govern proper coordination among shelterin, CST, and telomerase have not yet been fully understood. Here, we establish a conserved SWSSS motif, located adjacent to the Lys242 SUMOylation site in the fission yeast shelterin subunit Tpz1, as a new functional regulatory element for telomere protection and telomere length homeostasis. The SWSSS motif works redundantly with Lys242 SUMOylation to promote binding of Stn1-Ten1 at telomere and sub-telomere regions to protect against single-strand annealing (SSA)-dependent telomere fusions, and to prevent telomerase accumulation at telomeres. In addition, we provide evidence that the SWSSS motif defines an unanticipated role of Tpz1 in limiting telomerase activation at telomeres to prevent uncontrolled telomere elongation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Telomerase/metabolismo , Homeostase do Telômero , Proteínas de Ligação a Telômeros/metabolismo , Telômero/enzimologia , Motivos de Aminoácidos , Sequência Conservada , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Regulação Fúngica da Expressão Gênica , Ligação Proteica , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Sumoilação , Telômero/genética , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/genéticaRESUMO
The maintenance of telomere length is critical to longevity and survival. Specifically, the failure to properly replicate, resect, and/or form appropriate telomeric structures drives telomere shortening and, in turn, genomic instability. The endonuclease CtIP is a DNA repair protein that is well-known to promote genome stability through the resection of endogenous DNA double-stranded breaks. Here, we describe a novel role for CtIP. We show that in the absence of CtIP, human telomeres shorten rapidly to non-viable lengths. This telomere dysfunction results in an accumulation of fusions, breaks, and frank telomere loss. Additionally, CtIP suppresses the generation of circular, extrachromosomal telomeric DNA. These latter structures appear to arise from arrested DNA replication forks that accumulate in the absence of CtIP. Hence, CtIP is required for faithful replication through telomeres via its roles at stalled replication tracts. Our findings demonstrate a new role for CtIP as a protector of human telomere integrity.
Assuntos
Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica , Proteínas Nucleares/metabolismo , Encurtamento do Telômero/genética , Telômero/metabolismo , Reparo do DNA por Junção de Extremidades , Replicação do DNA , DNA Circular/metabolismo , Endodesoxirribonucleases , Humanos , Telômero/enzimologia , Proteínas de Ligação a Telômeros/metabolismoRESUMO
G-quadruplex telomeric secondary structures represent natural replication fork barriers and must be resolved to permit efficient replication. Stabilization of telomeric G4 leads to telomere dysfunctions demonstrated by telomere shortening or damage, resulting in genome instability and apoptosis. Chemical compounds targeting G4 structures have been reported to induce telomere disturbance and tumor suppression. Here, virtual screening was performed in a natural compound library using PyRx to identify novel G4 ligands. Emodin was identified as one of the best candidates, showing a great G4-binding potential. Subsequently, we confirmed that emodin could stabilize G4 structures in vitro and trigger telomere dysfunctions including fragile telomeres, telomere loss, and telomeric DNA damage. However, this telomere disturbance could be rescued by subsequent elevation of telomerase activity; in contrast, when we treated the cells with the telomerase inhibitor BIBR1532 upon emodin treatment, permanent telomere disturbance and obvious growth inhibition of 4T1-cell xenograft tumors were observed in mice. Taken together, our results show for the first time that emodin-induced telomeric DNA damage can upregulate telomerase activity, which may weaken its anticancer effect. The combined use of emodin and the telomerase inhibitor synergistically induced telomere dysfunction and inhibited tumor generation.
Assuntos
Aminobenzoatos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Emodina/uso terapêutico , Quadruplex G/efeitos dos fármacos , Naftalenos/uso terapêutico , Telomerase/antagonistas & inibidores , Encurtamento do Telômero/efeitos dos fármacos , Telômero/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Emodina/química , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/genética , Telomerase/metabolismo , Telômero/química , Telômero/enzimologia , Telômero/patologia , Transplante HeterólogoRESUMO
Overexpression of telomerase is one of the hallmarks of human cancer. Telomerase is important for maintaining the integrity of the ends of chromosomes, which are called telomeres. A growing number of human disease syndromes are associated with organ failure caused by mutations in telomerase (hTERT or hTR). Mutations in telomerase lead to telomere shortening by decreasing the stability of the telomerase complex, reducing its accumulation, or directly affecting its enzymatic activity. In this work, potential human telomerase mutations were identified by a systematic computational approach. Moreover, molecular docking methods were used to predict the effects of these mutations on the affinity of certain ligands (C_9i, C_9k, 16A, and NSC749234). The C_9k inhibitor had the best binding affinity for wild-type (WT) telomerase. Moreover, C_9i and C_9k had improved interactions with human telomerase in most of the mutant models. The R631 and Y717 residues of WT telomerase formed interactions with all studied ligands and these interactions were also commonly found in most of the mutant models. Residues forming stable interactions with ligands in molecular dynamics (MD) were traced, and the MD simulations showed that the C_9k ligand formed different conformations with WT telomerase than the C_9i ligand.
Assuntos
Mutação Puntual , Telomerase/genética , Encurtamento do Telômero/genética , Telômero/genética , Sítios de Ligação , Domínio Catalítico , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Telomerase/química , Telomerase/metabolismo , Telômero/enzimologia , Telômero/metabolismoRESUMO
Chromosome stability relies on an adequate length and complete replication of telomeres, the physical ends of chromosomes. Telomeres are composed of short direct repeat DNA and the associated nucleoprotein complex is essential for providing end-stability. In addition, the so-called end-replication problem of the conventional replication requires that telomeres be elongated by a special mechanism which, in virtually all organisms, is based by a reverse transcriptase, called telomerase. Although, at the conceptual level, telomere functions are highly similar in most organisms, the telomeric nucleoprotein composition appears to diverge significantly, in particular if it is compared between mammalian and budding yeast cells. However, over the last years, the CST complex has emerged as a central hub for telomere replication in most systems. Composed of three proteins, it is related to the highly conserved replication protein A complex, and in all systems studied, it coordinates telomerase-based telomere elongation with lagging-strand DNA synthesis. In budding yeast, the Cdc13 protein of this complex also is essential for telomerase recruitment and this specialisation is accompanied by additional regulatory adaptations. Based on recent results obtained in yeast, here, we review these issues and present an updated telomere replication hypothesis. We speculate that the similarities between systems far outweigh the differences, once we detach ourselves from the historic descriptions of the mechanisms in the various organisms.
Assuntos
Instabilidade Cromossômica/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Ligação a Telômeros/genética , Telômero/genética , Replicação do DNA/genética , Humanos , Modelos Genéticos , Mutação , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Telomerase/genética , Telomerase/metabolismo , Telômero/enzimologia , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Besides controlling epithelial-to-mesenchymal transition (EMT) and cell invasion, the Snail1 transcriptional factor also provides cells with cancer stem cell features. Since telomere maintenance is essential for stemness, we have examined the control of telomere integrity by Snail1. Fluorescence in situ hybridization (FISH) analysis indicates that Snail1-depleted mouse mesenchymal stem cells (MSC) have both a dramatic increase of telomere alterations and shorter telomeres. Remarkably, Snail1-deficient MSC present higher levels of both telomerase activity and the long non-coding RNA called telomeric repeat-containing RNA (TERRA), an RNA that controls telomere integrity. Accordingly, Snail1 expression downregulates expression of the telomerase gene (TERT) as well as of TERRA 2q, 11q and 18q. TERRA and TERT are transiently downregulated during TGFß-induced EMT in NMuMG cells, correlating with Snail1 expression. Global transcriptome analysis indicates that ectopic expression of TERRA affects the transcription of some genes induced during EMT, such as fibronectin, whereas that of TERT does not modify those genes. We propose that Snail1 repression of TERRA is required not only for telomere maintenance but also for the expression of a subset of mesenchymal genes.
Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição da Família Snail/genética , Homeostase do Telômero/genética , Telômero/genética , Animais , Linhagem Celular , Células Cultivadas , Transição Epitelial-Mesenquimal/genética , Perfilação da Expressão Gênica/métodos , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos Knockout , Células-Tronco Neoplásicas/metabolismo , RNA Longo não Codificante/genética , Fatores de Transcrição da Família Snail/metabolismo , Telomerase/genética , Telomerase/metabolismo , Telômero/enzimologiaRESUMO
BACKGROUND: Aging is a known risk factor of idiopathic pulmonary fibrosis (IPF). However, the pathogenic mechanisms underlying the effects of advanced aging remain largely unknown. Telomeric repeat-containing RNA (TERRA) represents a type of long noncoding RNA. In this study, the regulatory roles of TERRA on human telomeres and mitochondria and IPF epithelial injury model were identified. METHODS: Blood samples were collected from patients with IPF (n = 24) and matched control individuals (n = 24). The significance of clinical research on the TERRA expression correlated with pulmonary fibrosis was assessed. The expression levels of TERRA in vivo and in vitro were determined through quantitative real-time polymerase chain reaction analysis. Telomerase activity was observed using a fluorescent quantitative TRAP assay kit. The functions of telomeres, mitochondria, and associated genes were analyzed through RNA interference on TERRA. RESULTS: TERRA expression levels significantly increased in the peripheral blood mononuclear cells of IPF patients. The expression levels also exhibited a direct and significantly inverse correlation with the percentage of predicted force vital capacity, which is a physiological indicator of fibrogenesis during IPF progression. This finding was confirmed in the epithelial injury model of IPF in vitro. RNA interference on TERRA expression can ameliorate the functions of telomeres; mitochondria; associated genes; components associated with telomeres, such as telomerase reverse transcriptase, telomerase, and cell nuclear antigen, cyclin D1; and mitochondria-associated cyclin E genes, including the MMP and Bcl-2 family. The RNA interference on TERRA expression can also improve the functions of oxidative-stress-associated genes, such as reactive oxygen species, superoxide dismutase, and catalase, and apoptosis-related genes, such as cytochrome c, caspase-9, and caspase-3. CONCLUSIONS: In this study, the regulation of TERRA expression on telomeres and mitochondria during IPF pathogenesis was identified for the first time. The results may provide valuable insights for the discovery of a novel biomarker or therapeutic approach for IPF treatment.
Assuntos
Envelhecimento/genética , Fibrose Pulmonar Idiopática/genética , Mitocôndrias/enzimologia , RNA Longo não Codificante/genética , Telomerase/metabolismo , Telômero/enzimologia , Telômero/genética , Células A549/fisiologia , Células A549/ultraestrutura , Idoso , Animais , Apoptose/efeitos dos fármacos , Estudos de Casos e Controles , Catalase/metabolismo , Proliferação de Células , Feminino , Humanos , Peróxido de Hidrogênio/farmacologia , Fibrose Pulmonar Idiopática/sangue , Fibrose Pulmonar Idiopática/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/ultraestrutura , Interferência de RNA , RNA Longo não Codificante/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Homeostase do Telômero , Proteína Supressora de Tumor p53/genética , Capacidade Vital/genéticaRESUMO
Telomeres are highly susceptible to oxidative DNA damage, which if left unrepaired can lead to dysregulation of telomere length homeostasis. Here we employed single molecule FRET, single molecule pull-down and biochemical analysis to investigate how the most common oxidative DNA lesions, 8-oxoguanine (8oxoG) and thymine glycol (Tg), regulate the structural properties of telomeric DNA and telomerase extension activity. In contrast to 8oxoG which disrupts the telomeric DNA structure, Tg exhibits substantially reduced perturbation of G-quadruplex folding. As a result, 8oxoG induces high accessibility, whereas Tg retains limited accessibility, of telomeric G-quadruplex DNA to complementary single stranded DNA and to telomere binding protein POT1. Surprisingly, the Tg lesion stimulates telomerase loading and activity to a similar degree as an 8oxoG lesion. We demonstrate that this unexpected stimulation arises from Tg-induced conformational alterations and dynamics in telomeric DNA. Despite impacting structure by different mechanisms, both 8oxoG and Tg enhance telomerase binding and extension activity to the same degree, potentially contributing to oncogenesis.
Assuntos
Dano ao DNA , Estresse Oxidativo , Telomerase/metabolismo , Telômero/enzimologia , Sequência de Bases , DNA/química , DNA/genética , DNA/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Quadruplex G , Guanina/análogos & derivados , Guanina/química , Guanina/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Complexo Shelterina , Telômero/genética , Homeostase do Telômero/genética , Proteínas de Ligação a Telômeros/metabolismo , Timina/análogos & derivados , Timina/química , Timina/metabolismoRESUMO
AURKB (Aurora Kinase B) is a serine/threonine kinase better known for its role at the mitotic kinetochore during chromosome segregation. Here, we demonstrate that AURKB localizes to the telomeres in mouse embryonic stem cells, where it interacts with the essential telomere protein TERF1. Loss of AURKB function affects TERF1 telomere binding and results in aberrant telomere structure. In vitro kinase experiments successfully identified Serine 404 on TERF1 as a putative AURKB target site. Importantly, in vivo overexpression of S404-TERF1 mutants results in fragile telomere formation. These findings demonstrate that AURKB is an important regulator of telomere structural integrity.
Assuntos
Aurora Quinase B/metabolismo , Telômero/enzimologia , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Animais , Aurora Quinase B/fisiologia , Linhagem Celular , Células-Tronco Embrionárias/enzimologia , Humanos , Interfase/genética , Camundongos , Mitose/genética , Mutação , Ligação Proteica , Telômero/ultraestrutura , Proteína 1 de Ligação a Repetições Teloméricas/química , Proteína 1 de Ligação a Repetições Teloméricas/genéticaRESUMO
Cancer cells maintain telomere length equilibrium to avoid senescence and apoptosis induced by short telomeres, which trigger the DNA damage response. Limiting the potential for telomere maintenance in cancer cells has been long been proposed as a therapeutic target. Using an unbiased shRNA screen targeting known kinases, we identified bromodomain-containing protein 4 (BRD4) as a telomere length regulator. Four independent BRD4 inhibitors blocked telomere elongation, in a dose-dependent manner, in mouse cells overexpressing telomerase. Long-term treatment with BRD4 inhibitors caused telomere shortening in both mouse and human cells, suggesting BRD4 plays a role in telomere maintenance in vivo. Telomerase enzymatic activity was not directly affected by BRD4 inhibition. BRD4 is in clinical trials for a number of cancers, but its effects on telomere maintenance have not been previously investigated.
Assuntos
Proteínas Nucleares/genética , Homeostase do Telômero/genética , Encurtamento do Telômero/genética , Fatores de Transcrição/genética , Acetanilidas/farmacologia , Animais , Azepinas/farmacologia , Southern Blotting , Proteínas de Ciclo Celular , Linhagem Celular , Relação Dose-Resposta a Droga , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Expressão Gênica/efeitos dos fármacos , Células HeLa , Compostos Heterocíclicos com 3 Anéis/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Hibridização in Situ Fluorescente , Camundongos , Morfolinas/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Pironas/farmacologia , Interferência de RNA , Telomerase/genética , Telomerase/metabolismo , Telômero/efeitos dos fármacos , Telômero/enzimologia , Telômero/genética , Homeostase do Telômero/efeitos dos fármacos , Encurtamento do Telômero/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Triazóis/farmacologiaRESUMO
This literature review presents results of research showing association between functional activity of the telomere-telomerase system and mental cognitive and emotional processes in normal and various pathological states: chronic stress, depression, bipolar disorder, schizophrenia, mild cognitive impairment and dementia in aging. It also refers to age-specific, psycho-social, economic, immunological, genetic and epigenetic factors that influence these relationships.
Assuntos
Envelhecimento/fisiologia , Cognição/fisiologia , Emoções/fisiologia , Telomerase/fisiologia , Telômero/fisiologia , Transtorno Bipolar/etiologia , Disfunção Cognitiva/etiologia , Demência/etiologia , Depressão/etiologia , Humanos , Pesquisa , Esquizofrenia/etiologia , Telômero/enzimologiaRESUMO
The role of telomere shortening in the induction of replicative cellular senescence (CS) is well known and as a result, the involvement of telomerase and in particular its catalytic subunit, the telomerase reverse transcriptase (TERT) in CS has also been investigated. However, the majority of studies were conducted on cells that generally express high levels of TERT (cancer and immortalized cells) while the role of telomerase in CS in normal cells has been investigated to a much lesser extent. In particular, it was reported that active TERT is expressed in early passages of cultured human keratinocytes but rapidly diminished towards entry to CS, without telomere shortening. With the putative importance of TERT/telomerase in CS and the aging process in mind, we investigated the expression of TERT and telomerase activity in primary cultures of adult human dermal fibroblasts (HDFs) in the in vitro model of replicative CS. We found that (i) HDFs expressed active TERT; (ii) TERT protein levels and telomerase activity were markedly decreased in senescent HDFs; and (iii) the reduction of TERT in the soluble fraction was more pronounced than in the DNA-bound one. The results suggest the importance of the non-canonical (telomere-unrelated) functions of TERT in cellular senescence.
Assuntos
Senescência Celular , DNA/metabolismo , Fibroblastos/enzimologia , Telomerase/metabolismo , Encurtamento do Telômero , Telômero/enzimologia , Proliferação de Células , Células Cultivadas , Regulação para Baixo , Humanos , Solubilidade , Fatores de TempoRESUMO
Telomeric repeat binding factor 1 (TRF1) is essential to the maintenance of telomere chromatin structure and integrity. However, how telomere integrity is maintained, especially in response to damage, remains poorly understood. Here, we identify Nek7, a member of the Never in Mitosis Gene A (NIMA) kinase family, as a regulator of telomere integrity. Nek7 is recruited to telomeres and stabilizes TRF1 at telomeres after damage in an ATM activation-dependent manner. Nek7 deficiency leads to telomere aberrations, long-lasting γH2AX and 53BP1 foci, and augmented cell death upon oxidative telomeric DNA damage. Mechanistically, Nek7 interacts with and phosphorylates TRF1 on Ser114, which prevents TRF1 from binding to Fbx4, an Skp1-Cul1-F box E3 ligase subunit, thereby alleviating proteasomal degradation of TRF1, leading to a stable association of TRF1 with Tin2 to form a shelterin complex. Our data reveal a mechanism of efficient protection of telomeres from damage through Nek7-dependent stabilization of TRF1.
Assuntos
Dano ao DNA , Quinases Relacionadas a NIMA/metabolismo , Estresse Oxidativo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/enzimologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sítios de Ligação , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Quinases Relacionadas a NIMA/genética , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estabilidade Proteica , Interferência de RNA , Complexo Shelterina , Telômero/genética , Telômero/efeitos da radiação , Proteínas de Ligação a Telômeros/genética , Fatores de Tempo , Transfecção , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , UbiquitinaçãoRESUMO
Oxidative DNA damage triggers telomere erosion and cellular senescence. However, how repair is initiated at telomeres is largely unknown. Here, we found unlike PARP1-mediated Poly-ADP-Ribosylation (PARylation) at genomic damage sites, PARylation at telomeres is mainly dependent on tankyrase1 (TNKS1). TNKS1 is recruited to damaged telomeres via its interaction with TRF1, which subsequently facilitates the PARylation of TRF1 after damage. TNKS inhibition abolishes the recruitment of the repair proteins XRCC1 and polymerase ß at damaged telomeres, while the PARP1/2 inhibitor only has such an effect at non-telomeric damage sites. The ANK domain of TNKS1 is essential for the telomeric damage response and TRF1 interaction. Mutation of the tankyrase-binding motif (TBM) on TRF1 (13R/18G to AA) disrupts its interaction with TNKS1 concomitant recruitment of TNKS1 and repair proteins after damage. Either TNKS1 inhibition or TBM mutated TRF1 expression markedly sensitizes cells to telomere oxidative damage as well as XRCC1 inhibition. Together, our data reveal a novel role of TNKS1 in facilitating SSBR at damaged telomeres through PARylation of TRF1, thereby protecting genome stability and cell viability.