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1.
Biochem Soc Trans ; 49(5): 1927-1939, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34623385

RESUMO

Telomerase ribonucleoprotein was discovered over three decades ago as a specialized reverse transcriptase that adds telomeric repeats to the ends of linear eukaryotic chromosomes. Telomerase plays key roles in maintaining genome stability; and its dysfunction and misregulation have been linked to different types of cancers and a spectrum of human genetic disorders. Over the years, a wealth of genetic and biochemical studies of human telomerase have illuminated its numerous fascinating features. Yet, structural studies of human telomerase have lagged behind due to various challenges. Recent technical developments in cryo-electron microscopy have allowed for the first detailed visualization of the human telomerase holoenzyme, revealing unprecedented insights into its active site and assembly. This review summarizes the cumulative work leading to the recent structural advances, as well as highlights how the future structural work will further advance our understanding of this enzyme.


Assuntos
Telomerase/química , Telomerase/metabolismo , Biocatálise , Domínio Catalítico , Microscopia Crioeletrônica/métodos , Disceratose Congênita/enzimologia , Disceratose Congênita/genética , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Humanos , Modelos Moleculares , Mutação , Telomerase/genética , Telômero/metabolismo , Homeostase do Telômero
2.
Proteins ; 82(6): 897-903, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24130156

RESUMO

Several studies have recently shown that germline mutations in RTEL1, an essential DNA helicase involved in telomere regulation and DNA repair, cause Hoyeraal-Hreidarsson syndrome (HHS), a severe form of dyskeratosis congenita. Using original new softwares, facilitating the delineation of the different domains of the protein and the identification of remote relationships for orphan domains, we outline here that the C-terminal extension of RTEL1, downstream of its catalytic domain and including several HHS-associated mutations, contains a yet unidentified tandem of harmonin-N-like domains, which may serve as a hub for partner interaction. This finding highlights the potential critical role of this region for the function of RTEL1 and gives insights into the impact that the identified mutations would have on the structure and function of these domains.


Assuntos
DNA Helicases/química , Disceratose Congênita/genética , Retardo do Crescimento Fetal/genética , Deficiência Intelectual/genética , Microcefalia/genética , Sequência de Aminoácidos , Sequência Conservada , DNA Helicases/genética , Disceratose Congênita/enzimologia , Retardo do Crescimento Fetal/enzimologia , Duplicação Gênica , Mutação em Linhagem Germinativa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Deficiência Intelectual/enzimologia , Microcefalia/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína
3.
Hum Genet ; 132(4): 473-80, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23329068

RESUMO

Dyskeratosis congenita (DC) is an inherited bone marrow failure and cancer predisposition syndrome caused by aberrant telomere biology. The classic triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia is diagnostic of DC, but substantial clinical heterogeneity exists; the clinically severe variant Hoyeraal Hreidarsson syndrome (HH) also includes cerebellar hypoplasia, severe immunodeficiency, enteropathy, and intrauterine growth retardation. Germline mutations in telomere biology genes account for approximately one-half of known DC families. Using exome sequencing, we identified mutations in RTEL1, a helicase with critical telomeric functions, in two families with HH. In the first family, two siblings with HH and very short telomeres inherited a premature stop codon from their mother who has short telomeres. The proband from the second family has HH and inherited a premature stop codon in RTEL1 from his father and a missense mutation from his mother, who also has short telomeres. In addition, inheritance of only the missense mutation led to very short telomeres in the proband's brother. Targeted sequencing identified a different RTEL1 missense mutation in one additional DC proband who has bone marrow failure and short telomeres. Both missense mutations affect the helicase domain of RTEL1, and three in silico prediction algorithms suggest that they are likely deleterious. The nonsense mutations both cause truncation of the RTEL1 protein, resulting in loss of the PIP box; this may abrogate an important protein-protein interaction. These findings implicate a new telomere biology gene, RTEL1, in the etiology of DC.


Assuntos
DNA Helicases/genética , Disceratose Congênita/genética , Mutação em Linhagem Germinativa , Mutação de Sentido Incorreto , Adulto , Criança , Pré-Escolar , DNA Helicases/metabolismo , Disceratose Congênita/enzimologia , Exoma , Família , Feminino , Humanos , Lactente , Masculino , Estrutura Terciária de Proteína , Telômero/enzimologia , Telômero/genética
4.
Int Ophthalmol ; 32(6): 615-22, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22814958

RESUMO

The aim of this study is to present the limbal stem cell deficiency (LSCD) cases with features resembling dyskeratosis congenita (DC), a heritable disease of stem cells principally caused by telomerase deficiency. The clinical, laboratory and molecular findings of four cases are presented. A complete systemic examination was performed in a standardized manner for each patient. Laboratory measurements included investigations of the tests used for screening DC. All eight known disease-causing genes in DC (DKC1, TERC, TERT, NOP10, NHP2, TINF2, C16orf57, and TCAB1) were screened for mutations. The family members of the cases were also assessed, when possible. In all four patients, multisystem involvement was present, along with the disorder affecting corneal LSCs. The affected tissues were mainly the skin and its adnexa, the oral cavity and the hematopoietic system, which are rapidly renewing tissues, consistent with the presence of a stem cell disorder. Similarly affected cases were seen in different generations in families, suggesting an underlying inherited disorder. No mutation was detected in any of the known disease-causing genes in these patients. Based on the presented cases and with the contribution of the review of previously reported DC cases available, we suggest that DC is one of the inherited causes of LSCD and that those cases presenting with LSCD might represent a subgroup of DC caused by mutations in an as yet undefined gene.


Assuntos
Disceratose Congênita/genética , Limbo da Córnea/patologia , Mutação , RNA/genética , Células-Tronco/patologia , Telomerase/genética , Adulto , Disceratose Congênita/diagnóstico , Disceratose Congênita/enzimologia , Feminino , Humanos , Masculino , Linhagem , RNA/metabolismo , Telomerase/metabolismo
5.
Hum Mol Genet ; 21(4): 721-9, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22058290

RESUMO

X-linked dyskeratosis congenita (X-DC) is caused by mutations in the housekeeping nucleolar protein dyskerin. Amino acid changes associated with X-DC are remarkably heterogeneous. Peripheral mononuclear blood cells and fibroblasts isolated from X-DC patients harbor lower steady-state telomerase RNA (TER) levels and shorter telomeres than healthy age-matched controls. Previously, we showed that retroviral expression of recombinant TER, together with expression of recombinant telomerase reverse transcriptase, restored telomere maintenance and proliferative capacity in X-DC patient cells. Using rare X-DC isoforms (ΔL37 and A386T dyskerin), we showed that telomere maintenance defects observed in X-DC are solely due to decreased steady-state levels of TER. Disease-associated reductions in steady-state TER levels cause deficiencies in telomere maintenance. Here, we confirm these findings in other primary X-DC patient cell lines coding for the most common (A353V dyskerin) and more clinically severe (K314R and A353V dyskerin) X-DC isoforms. Using cell lines derived from these patients, we also examined the steady-state levels of other hinge-ACA motif RNAs and did not find differences in their in vivo accumulations. We show, for the first time, that purified telomerase holoenzyme complexes from different X-DC cells have normal catalytic activity. Our data confirm that dyskerin promotes TER stability in vivo, endorsing the development of TER supplementation strategies for the treatment of X-DC.


Assuntos
Proteínas de Ciclo Celular/genética , Disceratose Congênita/genética , Disceratose Congênita/metabolismo , Proteínas Nucleares/genética , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo , Biocatálise , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Disceratose Congênita/enzimologia , Disceratose Congênita/patologia , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Expressão Gênica , Genótipo , Holoenzimas/isolamento & purificação , Holoenzimas/metabolismo , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Motivos de Nucleotídeos , RNA/genética , RNA/metabolismo , Estabilidade de RNA , Telomerase/genética , Telomerase/isolamento & purificação , Homeostase do Telômero
6.
Cell Cycle ; 11(1): 19-25, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22157096

RESUMO

Telomere length homeostasis is achieved by a balance of telomere shortening caused by DNA replication and nucleolytic attack and telomere lengthening by telomerase. The importance of telomere length maintenance to human health is best illustrated by dyskeratosis congenita (DC) a disease of telomere shortening caused by mutations in telomerase subunits. DC patients suffer stem cell depletion and die of bone marrow stem cell failure. Recently a new class of particularly severe DC patients was found to harbor mutations in the shelterin subunit TIN2. The DC-TIN2 mutations were clustered in small domain of unknown function. In a recently published study we showed that the DC mutation cluster in TIN2 harbored a binding site for heterochromatin protein 1 (HP1) and further, that HP1 binding to TIN2 was required for sister telomere cohesion in S phase and for telomere length maintenance by telomerase. We briefly review and discuss the implications of our findings in this Extra View, and present some new data that may shed light on how sister telomere cohesion could influence telomere elongation by telomerase.


Assuntos
Telomerase/metabolismo , Telômero/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Disceratose Congênita/enzimologia , Disceratose Congênita/metabolismo , Disceratose Congênita/patologia , Humanos , Mutação , Ligação Proteica , Fase S , Telomerase/genética , Telômero/genética , Homeostase do Telômero , Encurtamento do Telômero , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
7.
Mol Cell ; 44(4): 660-6, 2011 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-22099312

RESUMO

How pseudouridylation (Ψ), the most common and evolutionarily conserved modification of rRNA, regulates ribosome activity is poorly understood. Medically, Ψ is important because the rRNA Ψ synthase, DKC1, is mutated in X-linked dyskeratosis congenita (X-DC) and Hoyeraal-Hreidarsson (HH) syndrome. Here, we characterize ribosomes isolated from a yeast strain in which Cbf5p, the yeast homolog of DKC1, is catalytically impaired through a D95A mutation (cbf5-D95A). Ribosomes from cbf5-D95A cells display decreased affinities for tRNA binding to the A and P sites as well as the cricket paralysis virus internal ribosome entry site (IRES), which interacts with both the P and the E sites of the ribosome. This biochemical impairment in ribosome activity manifests as decreased translational fidelity and IRES-dependent translational initiation, which are also evident in mouse and human cells deficient for DKC1 activity. These findings uncover specific roles for Ψ modification in ribosome-ligand interactions that are conserved in yeast, mouse, and humans.


Assuntos
Proteínas de Ciclo Celular/deficiência , Disceratose Congênita/genética , Retardo do Crescimento Fetal/genética , Hidroliases/deficiência , Hidroliases/metabolismo , Deficiência Intelectual/genética , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Nucleares/deficiência , RNA Ribossômico/metabolismo , RNA de Transferência/metabolismo , Ribonucleoproteínas Nucleares Pequenas/deficiência , Saccharomyces cerevisiae/genética , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Disceratose Congênita/enzimologia , Retardo do Crescimento Fetal/enzimologia , Genes Reporter , Humanos , Hidroliases/genética , Deficiência Intelectual/enzimologia , Luciferases/análise , Camundongos , Microcefalia/enzimologia , Proteínas Associadas aos Microtúbulos/genética , Mutação , Proteínas Nucleares/genética , Plasmídeos , Biossíntese de Proteínas , RNA Ribossômico/química , RNA Ribossômico/genética , RNA de Transferência/química , RNA de Transferência/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Ribossomos/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Transdução Genética
8.
Genes Dev ; 25(1): 11-6, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21205863

RESUMO

Dyskeratosis congenita (DC) is a genetic disorder of defective tissue maintenance and cancer predisposition caused by short telomeres and impaired stem cell function. Telomerase mutations are thought to precipitate DC by reducing either the catalytic activity or the overall levels of the telomerase complex. However, the underlying genetic mutations and the mechanisms of telomere shortening remain unknown for as many as 50% of DC patients, who lack mutations in genes controlling telomere homeostasis. Here, we show that disruption of telomerase trafficking accounts for unknown cases of DC. We identify DC patients with missense mutations in TCAB1, a telomerase holoenzyme protein that facilitates trafficking of telomerase to Cajal bodies. Compound heterozygous mutations in TCAB1 disrupt telomerase localization to Cajal bodies, resulting in misdirection of telomerase RNA to nucleoli, which prevents telomerase from elongating telomeres. Our findings establish telomerase mislocalization as a novel cause of DC, and suggest that telomerase trafficking defects may contribute more broadly to the pathogenesis of telomere-related disease.


Assuntos
Disceratose Congênita/enzimologia , Disceratose Congênita/genética , Mutação/genética , Transporte Proteico/fisiologia , Telomerase/metabolismo , Sequência de Aminoácidos , Animais , Disceratose Congênita/fisiopatologia , Humanos , Modelos Moleculares , Chaperonas Moleculares , Linhagem , Transporte Proteico/genética , Alinhamento de Sequência , Telomerase/genética
9.
Nature ; 464(7286): 292-6, 2010 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-20164838

RESUMO

Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer degeneration of multiple tissues. Patient-specific induced pluripotent stem (iPS) cells represent invaluable in vitro models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, which is accompanied by induction of the telomerase reverse transcriptase gene (TERT). We investigated whether defects in telomerase function would limit derivation and maintenance of iPS cells from patients with DC. Here we show that reprogrammed DC cells overcome a critical limitation in telomerase RNA component (TERC) levels to restore telomere maintenance and self-renewal. We discovered that TERC upregulation is a feature of the pluripotent state, that several telomerase components are targeted by pluripotency-associated transcription factors, and that in autosomal dominant DC, transcriptional silencing accompanies a 3' deletion at the TERC locus. Our results demonstrate that reprogramming restores telomere elongation in DC cells despite genetic lesions affecting telomerase, and show that strategies to increase TERC expression may be therapeutically beneficial in DC patients.


Assuntos
Disceratose Congênita/genética , Células-Tronco Pluripotentes , Telômero/genética , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular , Reprogramação Celular/genética , Disceratose Congênita/enzimologia , Regulação Enzimológica da Expressão Gênica , Humanos , Camundongos , Proteínas Nucleares/genética , Células-Tronco Pluripotentes/enzimologia , RNA/genética , RNA/metabolismo , Deleção de Sequência/genética , Telomerase/genética , Telomerase/metabolismo , Regulação para Cima
10.
Carcinogenesis ; 31(1): 9-18, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19887512

RESUMO

Myriad genetic and epigenetic alterations are required to drive normal cells toward malignant transformation. These somatic events commandeer many signaling pathways that cooperate to endow aspiring cancer cells with a full range of biological capabilities needed to grow, disseminate and ultimately kill its host. Cancer genomes are highly rearranged and are characterized by complex translocations and regional copy number alterations that target loci harboring cancer-relevant genes. Efforts to uncover the underlying mechanisms driving genome instability in cancer have revealed a prominent role for telomeres. Telomeres are nucleoprotein structures that protect the ends of eukaryotic chromosomes and are particularly vulnerable due to progressive shortening during each round of DNA replication and, thus, a lifetime of tissue renewal places the organism at risk for increasing chromosomal instability. Indeed, telomere erosion has been documented in aging tissues and hyperproliferative disease states-conditions strongly associated with increased cancer risk. Telomere dysfunction can produce the opposing pathophysiological states of degenerative aging or cancer with the specific outcome dictated by the integrity of DNA damage checkpoint responses. In most advanced cancers, telomerase is reactivated and serves to maintain telomere length and emerging data have also documented the capacity of telomerase to directly regulate cancer-promoting pathways. This review covers the role of telomeres and telomerase in the biology of normal tissue stem/progenitor cells and in the development of cancer.


Assuntos
Neoplasias/genética , Telomerase/metabolismo , Telômero , Animais , Senescência Celular , Disceratose Congênita/enzimologia , Disceratose Congênita/genética , Mutação em Linhagem Germinativa , Humanos , Camundongos , Camundongos Knockout , Neoplasias/enzimologia , Telomerase/genética , Proteína Supressora de Tumor p53/metabolismo
11.
Exp Dermatol ; 19(3): 279-88, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19558498

RESUMO

Dyskeratosis congenita (DC) is characterized by the triad of reticulate skin pigmentation, nail dystrophy and leukoplakia. Epidermal atrophy, hair growth defects, bone marrow failure and increased risk of cancer are also common in DC patients. DC is caused by mutations in genes encoding for telomerase complex factors. Although there is an association of epidermal abnormalities with DC, epidermal cells from DC donors have not been previously characterized. We have isolated skin keratinocytes from affected members of a family with an autosomal dominant form of DC that is caused by a mutation in the RNA component of telomerase, TERC. Here, we demonstrate that, similar to DC fibroblasts from these donors, DC keratinocytes have short telomeres and a short lifespan. DC keratinocytes also exhibited impaired colony forming efficiency (CFE) and migration capacity. Exogenous expression of the reverse transcriptase (RT) component of telomerase, TERT, activated telomerase levels to half that of TERT expressing normal cells and maintained telomeres at a short length with concomitant extension of lifespan. Unlike fibroblasts, transduction of human papillomavirus type 16 E6/E7 genes into DC keratinocytes activated telomerase to half that of E6/E7 expressing normal cells, and robust proliferation was observed. While expression of TERC has no measurable effect on telomerase in fibroblasts, expression of TERC in keratinocytes upregulated telomerase activity and, rarely, allowed rescue of proliferative defects. Our results point to important differences between DC fibroblasts and keratinocytes and show, for the first time, that expression of TERC can increase the lifespan of primary human epithelial cells.


Assuntos
Disceratose Congênita/enzimologia , Disceratose Congênita/patologia , Queratinócitos/enzimologia , Queratinócitos/patologia , RNA/genética , Telomerase/genética , Telomerase/metabolismo , Adulto , Sequência de Bases , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Primers do DNA/genética , Disceratose Congênita/genética , Ativação Enzimática , Feminino , Fibroblastos/enzimologia , Fibroblastos/patologia , Expressão Gênica , Humanos , Técnicas In Vitro , Masculino , Mutação , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , RNA/metabolismo , Proteínas Repressoras/genética , Telômero/genética , Telômero/patologia , Transdução Genética , Regulação para Cima , Adulto Jovem
12.
Blood ; 114(11): 2236-43, 2009 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-19561322

RESUMO

Androgens have been used in the treatment of bone marrow failure syndromes without a clear understanding of their mechanism of action. Blood counts of patients with dyskeratosis congenita or aplastic anemia with mutations in telomerase genes can improve with androgen therapy. Here we observed that exposure in vitro of normal peripheral blood lymphocytes and human bone marrow-derived CD34(+) cells to androgens increased telomerase activity, coincident with higher TERT mRNA levels. Cells from patients who were heterozygous for telomerase mutations had low baseline telomerase activity, which was restored to normal levels by exposure to androgens. Estradiol had an effect similar to androgens on TERT gene expression and telomerase enzymatic activity. Tamoxifen abolished the effects of both estradiol and androgens on telomerase function, and letrozole, an aromatase inhibitor, blocked androgen effects on telomerase activity. Conversely, flutamide, an androgen receptor antagonist, did not affect androgen stimulation of telomerase. Down-regulation by siRNA of estrogen receptor-alpha (ER alpha), but not ER beta, inhibited estrogen-stimulated telomerase function. Our results provide a mechanism for androgen therapy in bone marrow failure: androgens appear to regulate telomerase expression and activity mainly by aromatization and through ER alpha. These findings have potential implications for the choice of current androgenic compounds and the development of future agents for clinical use.


Assuntos
Androgênios/farmacologia , Estradiol/farmacologia , Estrogênios/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/enzimologia , Mutação , Telomerase/biossíntese , Antagonistas de Androgênios/farmacologia , Antagonistas de Receptores de Andrógenos , Androgênios/uso terapêutico , Anemia Aplástica/tratamento farmacológico , Anemia Aplástica/enzimologia , Anemia Aplástica/genética , Inibidores da Aromatase/farmacologia , Disceratose Congênita/tratamento farmacológico , Disceratose Congênita/enzimologia , Disceratose Congênita/genética , Estradiol/uso terapêutico , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio , Estrogênios/uso terapêutico , Feminino , Flutamida/farmacologia , Células-Tronco Hematopoéticas/patologia , Heterozigoto , Humanos , Letrozol , Linfócitos/enzimologia , Masculino , Nitrilas/farmacologia , Receptores Androgênicos/metabolismo , Tamoxifeno/farmacologia , Telomerase/genética , Triazóis/farmacologia
13.
Mol Cell Biol ; 29(1): 229-40, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18936156

RESUMO

The Protection of telomeres 1 (POT1) protein is a single-stranded telomere binding protein that is essential for proper maintenance of telomere length. Disruption of POT1 function leads to chromosome instability and loss of cellular viability. Here, we show that targeted deletion of the mouse Pot1b gene results in increased apoptosis in highly proliferative tissues. In the setting of telomerase haploinsufficiency, loss of Pot1b results in depletion of germ cells and complete bone marrow failure due to increased apoptosis, culminating in premature death. Pot1b(-/-) mTR(+/-) hematopoietic progenitor and stem cells display markedly reduced survival potential in vitro. Accelerated telomere shortening, increased G overhang and elevated number of chromosome end-to-end fusions that initiate an ATR-dependent DNA damage response were also observed. These results indicate an essential role for Pot1b in the maintenance of genome integrity and the long-term viability of proliferative tissues in the setting of telomerase deficiency. Interestingly, these phenotypes closely resemble those found in the human disease dyskeratosis congenita (DC), an inherited syndrome characterized by bone marrow failure, hyperpigmentation, and nail dystrophy. We anticipate that this mouse will serve as a useful model to further understand the pathophysiology of DC.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/deficiência , Disceratose Congênita/genética , Deleção de Genes , Haploidia , Proteínas Serina-Treonina Quinases/metabolismo , Telomerase/deficiência , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Células da Medula Óssea/enzimologia , Células da Medula Óssea/patologia , Morte Celular , Proliferação de Células , Disceratose Congênita/enzimologia , Sistema Hematopoético/anormalidades , Sistema Hematopoético/enzimologia , Sistema Hematopoético/patologia , Camundongos , Camundongos Knockout , Conformação de Ácido Nucleico , Especificidade de Órgãos , Fenótipo , Análise de Sobrevida , Telomerase/metabolismo , Telômero/química , Telômero/metabolismo
14.
Blood ; 111(5): 2606-14, 2008 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-18057229

RESUMO

Dyskerin gene is mutated in patients with X-linked dyskeratosis congenita (X-DC), which results in greatly reduced levels of telomerase activity. A genetic suppressor element (GSE) termed GSE24-2 has been isolated in a screening for cisplatin resistance. GSE24-2-expressing cells presented impaired telomerase inhibition following in vitro exposure to chemotherapies, such as cisplatin, or telomerase inhibitors. The promoter of the telomerase component hTERT was constitutively activated in GSE24-2 cells in a c-myc expression-dependent manner. Deletion analyses and mutagenesis of the human c-myc promoter demonstrated that the target sequence for activation was the nuclease hypersensitive element-III (NHEIII) site located upstream to the P1 region of the promoter. Further, expression of GSE24-2 in cell lines derived from patients with X-DC and in VA13 cells induced increased hTERT RNA and hTR levels and recovery of telomerase activity. Finally, expression of GSE24-2 was able to rescue X-DC fibroblasts from premature senescence. These data demonstrate that this domain of dyskerin plays an important role in telomerase maintenance following cell insults such as cisplatin treatment, and in telomerase-defective cells in patients with X-DC. The expression of this dyskerin fragment has a dominant function in X-DC cells and could provide the basis for a therapeutic approach to this disease.


Assuntos
Motivos de Aminoácidos , Proteínas de Ciclo Celular/química , Disceratose Congênita/enzimologia , Proteínas Nucleares/química , Fragmentos de Peptídeos/química , Telomerase/deficiência , Telomerase/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Resistência a Medicamentos/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myc/genética , RNA/metabolismo , Relação Estrutura-Atividade , Telomerase/antagonistas & inibidores
15.
Blood ; 111(3): 1128-30, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-18042801

RESUMO

Heterozygous mutations in the telomerase components TERT, the reverse transcriptase, and TERC, the RNA template, cause autosomal dominant dyskeratosis congenita due to telomere shortening. Anticipation, whereby the disease severity increases in succeeding generations due to inheritance of shorter telomeres, is a feature of this condition. Here we describe 2 families in which 2 TERT mutations are segregating. Both families contain compound heterozygotes. In one case the proband is homozygous for a novel mutation causing a P704S substitution, while his father's second allele encodes an H412Y mutation. The proband in the second family has mutant alleles Y846C and H876Q. Transfection studies show codominant expression of the mutated alleles with no evidence of a dominant negative effect or of intragenic complementation. Thus in these families the expression of both TERT alleles and the inherited telomere length contribute to the clinical phenotype.


Assuntos
Disceratose Congênita/enzimologia , Disceratose Congênita/genética , Predisposição Genética para Doença/genética , Telomerase/genética , Adulto , Idoso de 80 Anos ou mais , Criança , Disceratose Congênita/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Linhagem , Telomerase/metabolismo
16.
Blood ; 110(13): 4198-205, 2007 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17785587

RESUMO

Dyskeratosis congenita (DC) is a multisystem bone marrow failure syndrome characterized by a triad of mucocutaneous abnormalities and an increased predisposition to malignancy. X-linked DC is due to mutations in DKC1, while heterozygous mutations in TERC (telomerase RNA component) and TERT (telomerase reverse transcriptase) have been found in autosomal dominant DC. Many patients with DC remain uncharacterized, particularly families displaying autosomal recessive (AR) inheritance. We have now identified novel homozygous TERT mutations in 2 unrelated consanguineous families, where the index cases presented with classical DC or the more severe variant, Hoyeraal-Hreidarsson (HH) syndrome. These TERT mutations resulted in reduced telomerase activity and extremely short telomeres. As these mutations are homozygous, these patients are predicted to have significantly reduced telomerase activity in vivo. Interestingly, in contrast to patients with heterozygous TERT mutations or hemizygous DKC1 mutations, these 2 homozygous TERT patients were observed to have higher-than-expected TERC levels compared with controls. Collectively, the findings from this study demonstrate that homozygous TERT mutations, resulting in a pure but severe telomerase deficiency, produce a phenotype of classical AR-DC and its severe variant, the HH syndrome.


Assuntos
Disceratose Congênita/genética , Mutação , Telomerase/genética , Disceratose Congênita/enzimologia , Saúde da Família , Homozigoto , Humanos , Fenótipo , Síndrome , Telomerase/deficiência
17.
Blood ; 109(2): 524-32, 2007 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-16990594

RESUMO

Human telomerase hTERC RNA serves as a template for the catalytic hTERT protein to synthesize telomere repeats at chromosome ends. We have recently shown that some patients with bone marrow failure syndromes are heterozygous carriers for hTERC or hTERT mutations. These sequence variations usually lead to a compromised telomerase function by haploinsufficiency. Here, we provide functional characterization of an additional 8 distinct hTERT sequence variants and 5 hTERC variants that have recently been identified in patients with dyskeratosis congenita (DC) or aplastic anemia (AA). Among the mutations, 2 are novel telomerase variants that were identified in our cohort of patients. Whereas most of the sequence variants modulate telomerase function by haploinsufficiency, 2 hTERC variants with sequence changes located within the template region appear to act in a dominant-negative fashion. Inherited telomerase gene mutations, therefore, operate by various mechanisms to shorten telomere lengths, leading to limited marrow stem cell reserve and renewal capacity in patients with hematologic disorders.


Assuntos
Anemia Aplástica/genética , Disceratose Congênita/genética , Mutação , RNA/genética , Telomerase/genética , Adulto , Anemia Aplástica/enzimologia , Estudos de Coortes , Disceratose Congênita/enzimologia , Deleção de Genes , Variação Genética , Heterozigoto , Humanos , Masculino , Linhagem
18.
Semin Hematol ; 43(3): 157-66, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16822458

RESUMO

Dyskeratosis congenita (DC) is a rare inherited multi-system disorder. Although DC is classically characterized by mucocutaneous features, the vast majority of patients develop hematologic abnormalities, and in its occult form the disease can present as aplastic anemia. The gene responsible for the X-linked form of the disease encodes a protein involved in ribosome biogenesis and in stabilizing the telomerase complex, while the autosomal dominant form is caused by mutations in the core RNA component of telomerase. It has been suggested that DC is primarily a disease of defective telomere maintenance. Premature shortening of telomeres resulting in a limited proliferative potential of stem cells would explain the pathology observed in DC, as the affected tissues are those that require constant renewal.


Assuntos
Disceratose Congênita , Sequência de Aminoácidos , Anemia Aplástica/genética , Disceratose Congênita/enzimologia , Disceratose Congênita/genética , Disceratose Congênita/patologia , Humanos , Dados de Sequência Molecular , Mutação , Telomerase/genética
19.
Artigo em Inglês | MEDLINE | ID: mdl-17381301

RESUMO

Small functional RNAs play essential roles in many biological processes. Regulating the level of these small RNAs can be as important as maintaining their function in cells. The telomerase RNA is maintained in cells at a steady-state level where small changes in concentration can have a profound impact on function. Cells that have half the level of the telomerase RNA cannot maintain telomeres through many cell divisions. People who are heterozygous for telomerase RNA mutations have the diseases dyskeratosis congenita and aplastic anemia, caused by short telomeres that result in loss of tissue renewal capacity. Mice heterozygous for telomerase RNA show haploinsufficiency in telomere length maintenance and also show loss of tissue renewal capacity. It is remarkable that small changes in the level of this functional RNA can have such profound effects in cells. This tight regulation highlights the importance of controlling the action of telomerase in cells.


Assuntos
RNA/metabolismo , Telomerase/metabolismo , Telômero/metabolismo , Anemia Aplástica/enzimologia , Anemia Aplástica/genética , Animais , Divisão Celular , Disceratose Congênita/enzimologia , Disceratose Congênita/genética , Feminino , Heterozigoto , Humanos , Masculino , Camundongos , Camundongos Knockout , RNA/genética , Telomerase/deficiência , Telomerase/genética , Telômero/genética
20.
Proc Natl Acad Sci U S A ; 102(47): 17119-24, 2005 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-16284252

RESUMO

Telomeres protect chromosome ends from end-to-end fusion and degradation. Loss of telomere function causes cell-cycle arrest or cell death. Autosomal dominant dyskeratosis congenita (AD DC), a rare inherited bone marrow failure syndrome, is caused by mutations in TERC, the RNA component of telomerase. Here, we studied the telomere dynamics over three generations in a 32-member extended family with AD DC due to a TERC gene deletion. Our analysis shows that peripheral blood cells from family members haploinsufficient for TERC have very short telomeres. Telomeres are equally short in all individuals carrying the TERC gene deletion irrespective of their age. Chromosome-specific telomere analysis distinguishing the parental origin of telomeres showed that in gene deletion carriers, paternal and maternal telomeres are similarly short and similar in length to those of the affected parent. In children of affected parents who have normal TERC genes, parental telomeres are again similar in length, but two generations appear to be necessary to fully restore normal telomere length. These results are consistent with a model in which telomerase preferentially acts on the shortest telomeres. When TERC is limiting, this preference leads to the accelerated shortening of longer telomeres. The limited amount of active telomerase in TERC RNA haploinsufficiency may not be able to maintain the minimal length of the increasing number of short telomeres. Thus, the number of cells with excessively short telomeres and the degree of residual telomerase activity may determine the onset of disease in patients with AD DC.


Assuntos
Disceratose Congênita/enzimologia , Disceratose Congênita/genética , Haploidia , RNA/genética , Telomerase/genética , Telômero/enzimologia , Telômero/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Antecipação Genética/genética , Células Cultivadas , Criança , Pré-Escolar , Disceratose Congênita/fisiopatologia , Feminino , Deleção de Genes , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , RNA/fisiologia , Telomerase/fisiologia
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