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1.
J Biol Chem ; 297(3): 101080, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34403696

RESUMO

TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation.


Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Moléculas de Adesão Celular/fisiologia , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Microscopia de Força Atômica/métodos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Ligação Proteica , Isoformas de Proteínas/metabolismo , Complexo Shelterina/metabolismo , Complexo Shelterina/fisiologia , Telômero/metabolismo , Proteínas de Ligação a Telômeros/fisiologia , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/fisiologia
2.
Genes Dev ; 31(6): 567-577, 2017 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-28381410

RESUMO

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/fisiologia
3.
Genes Dev ; 31(6): 578-589, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28381412

RESUMO

Telomeres are protected by shelterin, a six-subunit protein complex that represses the DNA damage response (DDR) at chromosome ends. Extensive data suggest that TRF2 in shelterin remodels telomeres into the t-loop structure, thereby hiding telomere ends from double-stranded break repair and ATM signaling, whereas POT1 represses ATR signaling by excluding RPA. An alternative protection mechanism was suggested recently by which shelterin subunits TRF1, TRF2, and TIN2 mediate telomeric chromatin compaction, which was proposed to minimize access of DDR factors. We performed superresolution imaging of telomeres in mouse cells after conditional deletion of TRF1, TRF2, or both, the latter of which results in the complete loss of shelterin. Upon removal of TRF1 or TRF2, we observed only minor changes in the telomere volume in most of our experiments. Upon codeletion of TRF1 and TRF2, the telomere volume increased by varying amounts, but even those samples exhibiting small changes in telomere volume showed DDR at nearly all telomeres. Upon shelterin removal, telomeres underwent 53BP1-dependent clustering, potentially explaining at least in part the apparent increase in telomere volume. Furthermore, chromatin accessibility, as determined by ATAC-seq (assay for transposase-accessible chromatin [ATAC] with high-throughput sequencing), was not substantially altered by shelterin removal. These results suggest that the DDR induced by shelterin removal does not require substantial telomere decompaction.


Assuntos
Dano ao DNA , Telômero/ultraestrutura , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/fisiologia , Animais , Células Cultivadas , Cromatina/fisiologia , Camundongos , Microscopia de Fluorescência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/fisiologia
4.
Recent Results Cancer Res ; 200: 61-79, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26376872

RESUMO

Telomeres form protective caps at the ends of linear chromosomes to prevent nucleolytic degradation, end-to-end fusion, irregular recombination, and chromosomal instability. Telomeres are composed of repetitive DNA sequences (TTAGGG)n in humans, that are bound by specialized telomere binding proteins. Telomeres lose capping function in response to telomere shortening, which occurs during each division of cells that lack telomerase activity-the enzyme that can synthesize telomeres de novo. Telomeres have a dual role in cancer: telomere shortening can lead to induction of chromosomal instability and to the initiation of tumors, however, initiated tumors need to reactivate telomerase in order to stabilize chromosomes and to gain immortal growth capacity. In this review, we summarize current knowledge on the role of telomeres in the maintenance of chromosomal stability and carcinogenesis.


Assuntos
Instabilidade Cromossômica , Neoplasias/genética , Telômero/fisiologia , Animais , Proliferação de Células , Humanos , Telomerase/fisiologia , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia
5.
Oncotarget ; 6(9): 7011-22, 2015 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-25749521

RESUMO

TNKS1BP1 was originally identified as an interaction protein of tankyrase 1, which belongs to the poly(ADP-ribose) polymerase (PARP) superfamily. PARP members play important roles for example in DNA repair, telomere stability and mitosis regulation. Although the TNKS1BP1 protein was considered to be a poly(ADP-ribosyl)ation acceptor of tankyrase 1, its function is still unknown. Here we firstly identified that TNKS1BP1 was up-regulated by ionizing radiation (IR) and the depletion of TNKS1BP1 significantly sensitized cancer cells to IR. Neutral comet assay, pulsed-field gel electrophoresis, and γH2AX foci analysis indicated that TNKS1BP1 is required for the efficient repair of DNA double-strand breaks (DSB). The TNKS1BP1 protein was demonstrated to interact with DNA-dependent protein kinase (DNA-PKcs) and poly(ADP-ribose) polymerase 1 (PARP-1), by co-immunoprecipitation analysis. Moreover, TNKS1BP1 was shown to promote the association of PARP-1 and DNA-PKcs. Overexpression of TNKS1BP1 induced the autophosphorylation of DNA-PKcs/Ser2056 in a PARP-1 dependent manner, which contributed to an increased capability of DNA DSB repair. Inhibition of PARP-1 blocked the TNKS1BP1-mediated DNA-PKcs autophosphorylation and attenuated the PARylation of DNA-PKcs. TNKS1BP1 is a newly described component of the DNA DSB repair machinery, which provides much more mechanistic evidence for the rationale of developing effective anticancer measures by targeting PARP-1 and DNA-PKcs.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteína Quinase Ativada por DNA/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Antineoplásicos/química , Ensaio Cometa , Dano ao DNA , Células HeLa , Humanos , Fosforilação , Poli(ADP-Ribose) Polimerase-1 , Radiação Ionizante , Serina/química , Tanquirases/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/genética
6.
Biochemistry ; 53(34): 5485-95, 2014 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-25115914

RESUMO

A growing body of literature suggests that the homologous recombination/repair (HR) pathway cooperates with components of the shelterin complex to promote both telomere maintenance and nontelomeric HR. This may be due to the ability of both HR and shelterin proteins to promote strand invasion, wherein a single-stranded DNA (ssDNA) substrate base pairs with a homologous double-stranded DNA (dsDNA) template displacing a loop of ssDNA (D-loop). Rad51 recombinase catalyzes D-loop formation during HR, and telomere repeat binding factor 2 (TRF2) catalyzes the formation of a telomeric D-loop that stabilizes a looped structure in telomeric DNA (t-loop) that may facilitate telomere protection. We have characterized this functional interaction in vitro using a fluorescent D-loop assay measuring the incorporation of Cy3-labeled 90-nucleotide telomeric and nontelomeric substrates into telomeric and nontelomeric plasmid templates. We report that preincubation of a telomeric template with TRF2 inhibits the ability of Rad51 to promote telomeric D-loop formation upon preincubation with a telomeric substrate. This suggests Rad51 does not facilitate t-loop formation and suggests a mechanism whereby TRF2 can inhibit HR at telomeres. We also report a TRF2 mutant lacking the dsDNA binding domain promotes Rad51-mediated nontelomeric D-loop formation, possibly explaining how TRF2 promotes nontelomeric HR. Finally, we report telomere repeat binding factor 1 (TRF1) promotes Rad51-mediated telomeric D-loop formation, which may facilitate HR-mediated replication fork restart and explain why TRF1 is required for efficient telomere replication.


Assuntos
Rad51 Recombinase/fisiologia , Telômero , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/fisiologia , Sequência de Bases , Ligação Competitiva , Catálise , Primers do DNA , DNA de Cadeia Simples/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Fluorescência , Técnicas In Vitro
7.
Medicina (B Aires) ; 74(1): 69-76, 2014.
Artigo em Espanhol | MEDLINE | ID: mdl-24561847

RESUMO

Telomerase is the enzyme responsible for the maintenance of telomere length by adding guanine-rich repetitive sequences. Its activity can be seen in gametes, stem cells and tumor cells. In human somatic cells the proliferative potential is limited, reaching senescence after 50-70 cell divisions, because the DNA polymerase is not able to copy the DNA at the ends of chromosomes. By contrast, in most tumor cells the replicative potential is unlimited due to the maintenance of the telomeric length given by telomerase. Telomeres have additional proteins that regulate the binding of telomerase, likewise telomerase associates, with a protein complex that regulates its activity. This work focuses on the structure and function of the telomere/telomerase complex and how changes in its behavior lead to the development of different diseases, mainly cancer. Development of inhibitors of the telomere/telomerase complex could be a target with promising possibilities.


Assuntos
Neoplasias/genética , Telomerase/genética , Telômero/fisiologia , Animais , Divisão Celular/fisiologia , Senescência Celular/genética , Humanos , Neoplasias/enzimologia , Telomerase/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/fisiologia
8.
Medicina (B.Aires) ; 74(1): 69-76, ene.-feb. 2014. ilus
Artigo em Espanhol | BINACIS | ID: bin-131967

RESUMO

La telomerasa es la enzima responsable del mantenimiento de la longitud de los telómeros mediante la adición de secuencias repetitivas ricas en guanina, y su actividad se observa principalmente en gametos, células madre y células tumorales. En las células somáticas humanas el potencial de proliferación es limitado, alcanzando la senescencia luego de 50-70 divisiones celulares, debido a que la ADN polimerasa no es capaz de copiar el ADN en los extremos de los cromosomas. Por el contrario, en la mayoría de las células tumorales el potencial de replicación es ilimitado debido al mantenimiento de la longitud telomérica dado por la telomerasa. Los telómeros tienen proteínas adicionales que regulan la unión de la telomerasa. De la misma manera la telomerasa también se asocia con un complejo de proteínas que regulan su actividad. Este trabajo se centra en la estructura y función del complejo telómero/telomerasa y a cómo las alteraciones en su comportamiento conducen al desarrollo de diversas enfermedades, principalmente cáncer. El desarrollo de inhibidores del sistema telómero / telomerasa podría ser un blanco con posibilidades prometedoras.(AU)


Telomerase is the enzyme responsible for the maintenance of telomere length by adding guanine-rich repetitive sequences. Its activity can be seen in gametes, stem cells and tumor cells. In human somatic cells the proliferative potential is limited, reaching senescence after 50-70 cell divisions, because the DNA polymerase is not able to copy the DNA at the ends of chromosomes. By contrast, in most tumor cells the replicative potential is unlimited due to the maintenance of the telomeric length given by telomerase. Telomeres have additional proteins that regulate the binding of telomerase, likewise telomerase associates, with a protein complex that regulates its activity. This work focuses on the structure and function of the telomere/telomerase complex and how changes in its behavior lead to the development of different diseases, mainly cancer. Development of inhibitors of the telomere/telomerase complex could be a target with promising possibilities.(AU)


Assuntos
Animais , Humanos , Neoplasias/genética , Telomerase/genética , Telômero/fisiologia , Senescência Celular/genética , Divisão Celular/fisiologia , Neoplasias/enzimologia , Telomerase/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/fisiologia
9.
Medicina (B.Aires) ; 74(1): 69-76, ene.-feb. 2014. ilus
Artigo em Espanhol | LILACS | ID: lil-708560

RESUMO

La telomerasa es la enzima responsable del mantenimiento de la longitud de los telómeros mediante la adición de secuencias repetitivas ricas en guanina, y su actividad se observa principalmente en gametos, células madre y células tumorales. En las células somáticas humanas el potencial de proliferación es limitado, alcanzando la senescencia luego de 50-70 divisiones celulares, debido a que la ADN polimerasa no es capaz de copiar el ADN en los extremos de los cromosomas. Por el contrario, en la mayoría de las células tumorales el potencial de replicación es ilimitado debido al mantenimiento de la longitud telomérica dado por la telomerasa. Los telómeros tienen proteínas adicionales que regulan la unión de la telomerasa. De la misma manera la telomerasa también se asocia con un complejo de proteínas que regulan su actividad. Este trabajo se centra en la estructura y función del complejo telómero/telomerasa y a cómo las alteraciones en su comportamiento conducen al desarrollo de diversas enfermedades, principalmente cáncer. El desarrollo de inhibidores del sistema telómero / telomerasa podría ser un blanco con posibilidades prometedoras.


Telomerase is the enzyme responsible for the maintenance of telomere length by adding guanine-rich repetitive sequences. Its activity can be seen in gametes, stem cells and tumor cells. In human somatic cells the proliferative potential is limited, reaching senescence after 50-70 cell divisions, because the DNA polymerase is not able to copy the DNA at the ends of chromosomes. By contrast, in most tumor cells the replicative potential is unlimited due to the maintenance of the telomeric length given by telomerase. Telomeres have additional proteins that regulate the binding of telomerase, likewise telomerase associates, with a protein complex that regulates its activity. This work focuses on the structure and function of the telomere/telomerase complex and how changes in its behavior lead to the development of different diseases, mainly cancer. Development of inhibitors of the telomere/telomerase complex could be a target with promising possibilities.


Assuntos
Animais , Humanos , Neoplasias/genética , Telomerase/genética , Telômero/fisiologia , Senescência Celular/genética , Divisão Celular/fisiologia , Neoplasias/enzimologia , Telomerase/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , /fisiologia
10.
Medicina (B Aires) ; 74(1): 69-76, 2014.
Artigo em Espanhol | BINACIS | ID: bin-133732

RESUMO

Telomerase is the enzyme responsible for the maintenance of telomere length by adding guanine-rich repetitive sequences. Its activity can be seen in gametes, stem cells and tumor cells. In human somatic cells the proliferative potential is limited, reaching senescence after 50-70 cell divisions, because the DNA polymerase is not able to copy the DNA at the ends of chromosomes. By contrast, in most tumor cells the replicative potential is unlimited due to the maintenance of the telomeric length given by telomerase. Telomeres have additional proteins that regulate the binding of telomerase, likewise telomerase associates, with a protein complex that regulates its activity. This work focuses on the structure and function of the telomere/telomerase complex and how changes in its behavior lead to the development of different diseases, mainly cancer. Development of inhibitors of the telomere/telomerase complex could be a target with promising possibilities.


Assuntos
Neoplasias/genética , Telomerase/genética , Telômero/fisiologia , Animais , Senescência Celular/genética , Divisão Celular/fisiologia , Humanos , Neoplasias/enzimologia , Telomerase/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/fisiologia
11.
Blood ; 120(15): 2990-3000, 2012 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-22932806

RESUMO

TRF1 is part of the shelterin complex, which binds telomeres and it is essential for their protection. Ablation of TRF1 induces sister telomere fusions and aberrant numbers of telomeric signals associated with telomere fragility. Dyskeratosis congenita is characterized by a mucocutaneous triad, bone marrow failure (BMF), and presence of short telomeres because of mutations in telomerase. A subset of patients, however, show mutations in the shelterin component TIN2, a TRF1-interacting protein, presenting a more severe phenotype and presence of very short telomeres despite normal telomerase activity. Allelic variations in TRF1 have been found associated with BMF. To address a possible role for TRF1 dysfunction in BMF, here we generated a mouse model with conditional TRF1 deletion in the hematopoietic system. Chronic TRF1 deletion results in increased DNA damage and cellular senescence, but not increased apoptosis, in BM progenitor cells, leading to severe aplasia. Importantly, increased compensatory proliferation of BM stem cells is associated with rapid telomere shortening and further increase in senescent cells in vivo, providing a mechanism for the very short telomeres of human patients with mutations in the shelterin TIN2. Together, these results represent proof of principle that mutations in TRF1 lead to the main clinical features of BMF.


Assuntos
Medula Óssea/patologia , Senescência Celular , Modelos Animais de Doenças , Disceratose Congênita/etiologia , Sistema Hematopoético/patologia , Hemoglobinúria Paroxística/etiologia , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Anemia Aplástica , Animais , Apoptose , Doenças da Medula Óssea , Transtornos da Insuficiência da Medula Óssea , Transplante de Medula Óssea , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Dano ao DNA , Disceratose Congênita/patologia , Ensaio de Imunoadsorção Enzimática , Feminino , Hemoglobinúria Paroxística/mortalidade , Hemoglobinúria Paroxística/patologia , Humanos , Integrases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação/genética , Pancitopenia/etiologia , Pancitopenia/metabolismo , Pancitopenia/patologia , Células-Tronco/patologia , Taxa de Sobrevida , Telômero/genética , Proteínas de Ligação a Telômeros/genética
12.
Bull Cancer ; 97(9): 1087-104, 2010 Sep.
Artigo em Francês | MEDLINE | ID: mdl-20663741

RESUMO

Advances in chromosome dynamics have increased our understanding of the significant role of telomeres and telomerase in cancer. Telomerase is expressed in almost all cancer cells but is inactive in most normal somatic cells. Therefore, telomerase is an important target for the design of therapeutic agents that might have minimal side effects. Herein, we evaluate current approaches to telomerase/telomere-targeted therapy, discuss the benefits and disadvantages, and speculate on the future direction of telomerase inhibitors as cancer therapeutics.


Assuntos
Neoplasias/tratamento farmacológico , Interferência de RNA , Telomerase/antagonistas & inibidores , Telômero , Divisão Celular/fisiologia , Senescência Celular/fisiologia , Inibidores Enzimáticos/uso terapêutico , Previsões , Quadruplex G , Terapia Genética/métodos , Humanos , Imunoterapia/métodos , Proteínas de Neoplasias/fisiologia , Neoplasias/enzimologia , Neoplasias/genética , Oligonucleotídeos/uso terapêutico , Oligonucleotídeos Antissenso/uso terapêutico , RNA , Processamento Pós-Transcricional do RNA , RNA Catalítico/uso terapêutico , RNA Longo não Codificante , RNA não Traduzido/antagonistas & inibidores , Inibidores da Transcriptase Reversa/uso terapêutico , Telomerase/metabolismo , Telomerase/fisiologia , Telômero/química , Telômero/genética , Telômero/imunologia , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/fisiologia , Transcrição Gênica
13.
Cancer Res ; 70(5): 2041-52, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-20160025

RESUMO

Aurora-A, a conserved serine-threonine kinase, plays essential roles in mitosis. Aberrant upregulation of Aurora-A perturbs proper mitotic progression and results in a generation of multinucleated cells with centrosome amplification. The molecular mechanisms for these mitotic defects remain elusive. Here, we show that the overexpressed Aurora-A-induced mitotic defects depend on the telomeric protein TRF1. Live and fixed cell analyses revealed that Aurora-A overexpression in HeLa cells compromises chromosome biorientation, which leads to cytokinetic failure and tetraploidization with increased centrosome numbers. TRF1 depletion by small interfering RNAs or by tankyrase-1 overexpression suppresses Aurora-A-induced occurrence of unaligned chromosomes in metaphase, thus preventing the subsequent abnormalities. We found that Aurora-A binds and phosphorylates TRF1. When TRF1 knockdown cells are complemented with wild-type TRF1, Aurora-A-induced mitotic defects recur. By contrast, a TRF1 mutant that is not phosphorylatable by Aurora-A does not restore such Aurora-A-induced phenotype. We propose that TRF1 phosphorylation by excessive Aurora-A may provoke abnormal mitosis and chromosomal instability.


Assuntos
Mitose/fisiologia , Proteínas Serina-Treonina Quinases/biossíntese , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Aurora Quinases , Centrossomo/fisiologia , Cromossomos Humanos , Células HeLa , Humanos , Mitose/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , Tanquirases/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Transfecção
14.
J Cell Biol ; 185(5): 827-39, 2009 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19487455

RESUMO

Telomeric repeat binding factor 1 (TRF1) is a component of the multiprotein complex "shelterin," which organizes the telomere into a high-order structure. TRF1 knockout embryos suffer from severe growth defects without apparent telomere dysfunction, suggesting an obligatory role for TRF1 in cell cycle control. To date, the mechanism regulating the mitotic increase in TRF1 protein expression and its function in mitosis remains unclear. Here, we identify guanine nucleotide-binding protein-like 3 (GNL3L), a GTP-binding protein most similar to nucleostemin, as a novel TRF1-interacting protein in vivo. GNL3L binds TRF1 in the nucleoplasm and is capable of promoting the homodimerization and telomeric association of TRF1, preventing promyelocytic leukemia body recruitment of telomere-bound TRF1, and stabilizing TRF1 protein by inhibiting its ubiquitylation and binding to FBX4, an E3 ubiquitin ligase for TRF1. Most importantly, the TRF1 protein-stabilizing activity of GNL3L mediates the mitotic increase of TRF1 protein and promotes the metaphase-to-anaphase transition. This work reveals novel aspects of TRF1 modulation by GNL3L.


Assuntos
Proteínas de Ligação ao GTP/fisiologia , Mitose/fisiologia , Proteínas Nucleares/fisiologia , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Polaridade Celular , Dimerização , Proteínas F-Box/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Humanos , Camundongos , Mitose/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Telomerase/metabolismo , Telômero/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Ubiquitinação
15.
Mol Cell Biol ; 29(6): 1608-25, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19124610

RESUMO

TRF1 is a component of the shelterin complex at mammalian telomeres; however, a role for TRF1 in telomere biology in the context of the organism is unclear. In this study, we generated mice with transgenic TRF1 expression targeted to epithelial tissues (K5TRF1 mice). K5TRF1 mice have shorter telomeres in the epidermis than wild-type controls do, and these are rescued in the absence of the XPF nuclease, indicating that TRF1 acts as a negative regulator of telomere length by controlling XPF activity at telomeres, similar to what was previously described for TRF2-overexpressing mice (K5TRF2 mice). K5TRF1 cells also show increased end-to-end chromosomal fusions, multitelomeric signals, and increased telomere recombination, indicating an impact of TRF1 on telomere integrity, again similar to the case in K5TRF2 cells. Intriguingly, K5TRF1 cells, but not K5TRF2 cells, show increased mitotic spindle aberrations. TRF1 colocalizes with the spindle assembly checkpoint proteins BubR1 and Mad2 at mouse telomeres, indicating a link between telomeres and the mitotic spindle. Together, these results demonstrate that TRF1, like TRF2, negatively regulates telomere length in vivo by controlling the action of the XPF nuclease at telomeres; in addition, TRF1 has a unique role in the mitotic spindle checkpoint.


Assuntos
Dano ao DNA/fisiologia , Epiderme/fisiologia , Queratinócitos/fisiologia , Telômero/fisiologia , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Animais , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Células Epidérmicas , Epitélio/fisiologia , Homeostase , Queratinócitos/citologia , Proteínas Mad2 , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/fisiologia , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo
16.
Cytometry A ; 75(5): 428-39, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19097172

RESUMO

Telomeres are complex end structures that confer functional integrity and positional stability to human chromosomes. Despite their critical importance, there is no clear view on telomere organization in cycling human cells and their dynamic behavior throughout the cell cycle. We investigated spatiotemporal organization of telomeres in living human ECV-304 cells stably expressing telomere binding proteins TRF1 and TRF2 fused to mCitrine using four dimensional microscopy. We thereby made use of controlled light exposure microscopy (CLEM), a novel technology that strongly reduces photodamage by limiting excitation in parts of the image where full exposure is not needed. We found that telomeres share small territories where they dynamically associate. These territories are preferentially positioned at the interface of chromatin domains. TRF1 and TRF2 are abundantly present in these territories but not firmly bound. At the onset of mitosis, the bulk of TRF protein dissociates from telomere regions, territories disintegrate and individual telomeres become faintly visible. The combination of stable cell lines, CLEM and cytometry proved essential in providing novel insights in compartment-based nuclear organization and may serve as a model approach for investigating telomere-driven genome-instability and studying long-term nuclear dynamics.


Assuntos
Ciclo Celular/fisiologia , Telômero/fisiologia , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/fisiologia , Células HeLa , Humanos , Microscopia de Fluorescência , Proteínas Recombinantes de Fusão/fisiologia , Transfecção
17.
Plant J ; 55(4): 709-17, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18466302

RESUMO

SUMMARY: The C(18) ketone (5E,7E)-6-methyl-8-(2,6,6-trimethylcyclohex-1-enyl)octa-5,7-dien-2-one (D'orenone) has been postulated to be an early cleavage product of beta-carotene en route to trisporic acids; these act as morphogenetic factors during the sexual reproduction of zygomycetes. Here we report that D'orenone blocks the highly polarized tip growth of root hairs, causing tip growth to stop completely within a few minutes. Importantly, external auxin reverses the effects of D'orenone on root hairs. Further analysis revealed that D'orenone lowers the auxin concentration in trichoblasts via PIN2-mediated auxin efflux to below the critical levels essential for root hair growth. D'orenone specifically increases PIN2 protein abundance without affecting PIN2 transcripts, and the PIN2 expression domain enlarges and shifts basipetally, resulting in more active auxin transport. The observation that D'orenone does not interfere with the root hair growth in roots of null mutant lines provides additional evidence that PIN2 is its specific target.


Assuntos
Ácidos Indolacéticos/metabolismo , Cetonas/farmacologia , Coifa/fisiologia , Raízes de Plantas/crescimento & desenvolvimento , Arabidopsis/efeitos dos fármacos , Arabidopsis/fisiologia , Dimetil Sulfóxido/farmacologia , Ácidos Indolacéticos/farmacologia , Coifa/citologia , Coifa/efeitos dos fármacos , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Proteína 1 de Ligação a Repetições Teloméricas/efeitos dos fármacos , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia
18.
J Biol Chem ; 283(11): 6935-41, 2008 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-18178559

RESUMO

Mammalian telomeres are composed of G-rich repetitive double-stranded (ds) DNA with a 3' single-stranded (ss) overhang and associated proteins that together maintain chromosome end stability. Complete replication of telomeric DNA requires de novo elongation of the ssDNA by the enzyme telomerase, with telomeric proteins playing a key role in regulating telomerase-mediated telomere replication. In regards to the protein component of mammalian telomeres, TRF1 and TRF2 bind to the dsDNA of telomeres, whereas POT1 binds to the ssDNA portion. These three proteins are linked through either direct interactions or by the proteins TIN2 and TPP1. To determine the biological consequence of connecting telomeric dsDNA to ssDNA through a multiprotein assembly, we compared the effect of expressing TRF1 and POT1 in trans versus in cis in the form of a fusion of these two proteins, on telomere length in telomerase-positive cells. When expressed in trans these two proteins induced extensive telomere elongation. Fusing TRF1 to POT1 abrogated this effect, inducing mild telomere shortening, and generated looped DNA structures, as assessed by electron microscopy, consistent with the protein forming a complex with dsDNA and ssDNA. We speculate that such a protein bridge between dsDNA and ssDNA may inhibit telomerase access, promoting telomere shortening.


Assuntos
DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , DNA/química , Regulação da Expressão Gênica , Proteínas Nucleares/fisiologia , Proteínas de Ligação a Telômeros/fisiologia , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/fisiologia , Linhagem Celular , Humanos , Microscopia de Fluorescência , Modelos Biológicos , Proteínas Nucleares/química , Conformação de Ácido Nucleico , Ligação Proteica , Complexo Shelterina , Telomerase/metabolismo , Telômero/ultraestrutura , Proteínas de Ligação a Telômeros/química , Proteína 1 de Ligação a Repetições Teloméricas/química , Proteína 2 de Ligação a Repetições Teloméricas/química , Tripeptidil-Peptidase 1
19.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 36(4): 325-30, 2007 07.
Artigo em Chinês | MEDLINE | ID: mdl-17717821

RESUMO

OBJECTIVE: To investigate the role of Ser 219 phosphorylation of TRF1 (telomere repeat binding factor 1) in regulation of cell cycle. METHODS: The mimicking phosphorylation mutant (TRF1S219D-GFP) and the non-phosphorylatable mutant (TRF1S219A-GFP) were constructed; the mutant genes and corresponding proteins were checked by sequencing and Western blot, respectively. Immunofluorescence staining was performed to detect the localization of mutants in HeLa cells. Cell cycle was analyzed by flow cytometry and ATM level was evaluated by immunoblotting. RESULTS: The mutant genes were verified by direct sequencing and protein expression of GFP-tagged mutants was confirmed by immunoblotting.TRF1S219A-GFP and TRF1S219D-GFP were both localized in telomere of HeLa cells. Moreover, overexpression of TRF1-GFP or TRF1S219A-GFP resulted in an accumulation of HeLa cells in G2/M (P<0.05). The protein level of ATM was increased when overexpression the wide type or mutants. CONCLUSION: The Ser 219 phosphorylation of TRF1 by ATM could result in cell cycle arrest in G2/M, which is related to overexpression of TRF1.


Assuntos
Ciclo Celular/fisiologia , Serina/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Immunoblotting , Microscopia de Fluorescência , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/genética , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Transfecção , Proteínas Supressoras de Tumor/metabolismo
20.
Nat Struct Mol Biol ; 14(9): 832-40, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17694070

RESUMO

Human telomeres are associated with ATM and the protein complex consisting of MRE11, RAD50 and NBS1 (MRN), which are central to maintaining genomic stability. Here we show that when targeted to telomeres, wild-type RAD50 downregulates telomeric association of TRF1, a negative regulator of telomere maintenance. TRF1 binding to telomeres is upregulated in cells deficient in NBS1 or under ATM inhibition. The TRF1 association with telomeres induced by ATM inhibition is abrogated in cells lacking MRE11 or NBS1, suggesting that MRN and ATM function in the same pathway controlling TRF1 binding to telomeres. The ability of TRF1 to interact with telomeric DNA in vitro is impaired by ATM-mediated phosphorylation. We propose that MRN is required for TRF1 phosphorylation by ATM and that such phosphorylation results in the release of TRF1 from telomeres, promoting telomerase access to the ends of telomeres.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Enzimas Reparadoras do DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Telômero , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Hidrolases Anidrido Ácido , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular , Humanos , Proteína Homóloga a MRE11 , Fosforilação , Ligação Proteica
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