Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 194
Filtrar
1.
Nat Commun ; 11(1): 4979, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020468

RESUMO

Cellular senescence is a known driver of carcinogenesis and age-related diseases, yet senescence is required for various physiological processes. However, the mechanisms and factors that control the negative effects of senescence while retaining its benefits are still elusive. Here, we show that the rasGAP SH3-binding protein 1 (G3BP1) is required for the activation of the senescent-associated secretory phenotype (SASP). During senescence, G3BP1 achieves this effect by promoting the association of the cyclic GMP-AMP synthase (cGAS) with cytosolic chromatin fragments. In turn, G3BP1, through cGAS, activates the NF-κB and STAT3 pathways, promoting SASP expression and secretion. G3BP1 depletion or pharmacological inhibition impairs the cGAS-pathway preventing the expression of SASP factors without affecting cell commitment to senescence. These SASPless senescent cells impair senescence-mediated growth of cancer cells in vitro and tumor growth in vivo. Our data reveal that G3BP1 is required for SASP expression and that SASP secretion is a primary mediator of senescence-associated tumor growth.


Assuntos
Senescência Celular/fisiologia , DNA Helicases/metabolismo , Neoplasias/patologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Células A549 , Animais , Carcinogênese , Linhagem Celular , Movimento Celular , Citocinas/metabolismo , DNA Helicases/antagonistas & inibidores , DNA Helicases/deficiência , Humanos , Inflamação , Camundongos , Neoplasias/metabolismo , Nucleotidiltransferases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/deficiência , RNA Helicases/antagonistas & inibidores , RNA Helicases/deficiência , Proteínas com Motivo de Reconhecimento de RNA/antagonistas & inibidores , Proteínas com Motivo de Reconhecimento de RNA/deficiência , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/metabolismo
2.
Mol Cell ; 79(4): 645-659.e9, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32692974

RESUMO

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.


Assuntos
Grânulos Citoplasmáticos/genética , DNA Helicases/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Biossíntese de Proteínas , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Estresse Fisiológico/genética , Regiões 5' não Traduzidas , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/patologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Feminino , Células HCT116 , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Espermatogônias/citologia , Espermatogônias/patologia , Testículo/citologia , Testículo/metabolismo
3.
Gene ; 754: 144859, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32535049

RESUMO

DNA topoisomerases essentially remove topological strains generated during DNA replication, transcription, DNA repair, and other cytogenetic processes. However, distinct expression level and prognostic significance of individual topoisomerase isoforms in gastric cancer (GC) remain largely unexplored. In this study, we utilized Oncomine and Kaplan-Meier plotter database to detect the mRNA expression level of individual topoisomerase isoforms as well as assess their prognostic significance in GC patients. With the exception of TOP3B and TOP2B, levels of all topoisomerase isoforms were found to be elevated in GC patients when compared to the normal tissues. Elevated expression of TOP1 and TOP1MT was relevant to longer overall survival (OS) in GC and gastric intestinal type adenocarcinoma (GITA) patients, but not in diffuse gastric adenocarcinoma (DFA) patients. Increased expression of TOP2A and TOP2B was related to better OS in GC, as well as in GITA and DFA patients. In contrast, increased expression TOP3A and TOP3B was associated with shorter OS in GC, as well as in GITA and DFA patients. We also applied the Tumor IMmune Estimation Resource (TIMER) tool to assess the correlations between distinct topoisomerase isoforms and the infiltrating immune cell landscape. Furthermore, we found that down-regulating the expression of TOP3A by shRNA significantly inhibited the proliferation and colony formation in GC cells compared to control shRNA treated cells. Thus our study lays the framework for utilizing topoisomerases in better understanding the complexity and heterogeneity of GC and for developing strategies for novel customized therapy in GC patients.


Assuntos
Adenocarcinoma/patologia , Biomarcadores Tumorais/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Neoplasias Gástricas/patologia , Adenocarcinoma/enzimologia , Biomarcadores Tumorais/genética , DNA Topoisomerases Tipo II/genética , Precursores Enzimáticos , Perfilação da Expressão Gênica , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Prognóstico , Neoplasias Gástricas/enzimologia , Taxa de Sobrevida
4.
Mol Pharmacol ; 98(3): 222-233, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32587095

RESUMO

DNA topoisomerase II (TOP2) is required for the unwinding and decatenation of DNA through the induction of an enzyme-linked double-strand break (DSB) in one DNA molecule and passage of another intact DNA duplex through the break. Anticancer drugs targeting TOP2 (TOP2 poisons) prevent religation of the DSB and stabilize a normally transient intermediate of the TOP2 reaction mechanism called the TOP2-DNA covalent complex. Subsequently, TOP2 remains covalently bound to each end of the enzyme-bridged DSB, which cannot be repaired until TOP2 is removed from the DNA. One removal mechanism involves the proteasomal degradation of the TOP2 protein, leading to the liberation of a protein-free DSB. Proteasomal degradation is often regulated by protein ubiquitination, and here we show that inhibition of ubiquitin-activating enzymes reduces the processing of TOP2A- and TOP2B-DNA complexes. Depletion or inhibition of ubiquitin-activating enzymes indicated that ubiquitination was required for the liberation of etoposide-induced protein-free DSBs and is therefore an important layer of regulation in the repair of TOP2 poison-induced DNA damage. TOP2-DNA complexes stabilized by etoposide were shown to be conjugated to ubiquitin, and this was reduced by inhibition or depletion of ubiquitin-activating enzymes. SIGNIFICANCE STATEMENT: There is currently great clinical interest in the ubiquitin-proteasome system and ongoing development of specific inhibitors. The results in this paper show that the therapeutic cytotoxicity of DNA topoisomerase II (TOP2) poisons can be enhanced through combination therapy with ubiquitin-activating enzyme inhibitors or by specific inhibition of the BMI/RING1A ubiquitin ligase, which would lead to increased cellular accumulation or persistence of TOP2-DNA complexes.


Assuntos
DNA Topoisomerases Tipo II/metabolismo , Nucleosídeos/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Sulfonamidas/farmacologia , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Linhagem Celular , DNA/metabolismo , DNA Topoisomerases Tipo II/química , Humanos , Células K562 , Proteínas de Ligação a Poli-ADP-Ribose/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos
5.
Mol Cell ; 78(6): 1166-1177.e6, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32497495

RESUMO

Human tumors with exonuclease domain mutations in the gene encoding DNA polymerase ε (POLE) have incredibly high mutation burdens. These errors arise in four unique mutation signatures occurring in different relative amounts, the etiologies of which remain poorly understood. We used CRISPR-Cas9 to engineer human cell lines expressing POLE tumor variants, with and without mismatch repair (MMR). Whole-exome sequencing of these cells after defined numbers of population doublings permitted analysis of nascent mutation accumulation. Unlike an exonuclease active site mutant that we previously characterized, POLE cancer mutants readily drive signature mutagenesis in the presence of functional MMR. Comparison of cell line and human patient data suggests that the relative abundance of mutation signatures partitions POLE tumors into distinct subgroups dependent on the nature of the POLE allele, its expression level, and MMR status. These results suggest that different POLE mutants have previously unappreciated differences in replication fidelity and mutagenesis.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Alelos , Linhagem Celular Tumoral , Reparo de Erro de Pareamento de DNA/fisiologia , Humanos , Mutagênese/genética , Mutação/genética , Neoplasias/genética , Neoplasias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo
6.
Nat Chem Biol ; 16(9): 955-963, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32451507

RESUMO

Diverse RNAs and RNA-binding proteins form phase-separated, membraneless granules in cells under stress conditions. However, the role of the prevalent mRNA methylation, m6A, and its binding proteins in stress granule (SG) assembly remain unclear. Here, we show that m6A-modified mRNAs are enriched in SGs, and that m6A-binding YTHDF proteins are critical for SG formation. Depletion of YTHDF1/3 inhibits SG formation and recruitment of mRNAs to SGs. Both the N-terminal intrinsically disordered region and the C-terminal m6A-binding YTH domain of YTHDF proteins are important for SG formation. Super-resolution imaging further reveals that YTHDF proteins appear to be in a super-saturated state, forming clusters that often reside in the periphery of or at the junctions between SG core clusters, and potentially promote SG formation by reducing the activation energy barrier and critical size for SG condensate formation. Our results suggest a new function of the m6A-binding YTHDF proteins in regulating SG formation.


Assuntos
Adenina/análogos & derivados , Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenina/metabolismo , Linhagem Celular Tumoral , Grânulos Citoplasmáticos/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Humanos , Metiltransferases/genética , Metiltransferases/metabolismo , Mutação , Estresse Oxidativo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Domínios Proteicos , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
7.
Nat Commun ; 11(1): 2104, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32355176

RESUMO

The response to DNA damage-stalled RNA polymerase II (RNAPIIo) involves the assembly of the transcription-coupled repair (TCR) complex on actively transcribed strands. The function of the TCR proteins CSB, CSA and UVSSA and the manner in which the core DNA repair complex, including transcription factor IIH (TFIIH), is recruited are largely unknown. Here, we define the assembly mechanism of the TCR complex in human isogenic knockout cells. We show that TCR is initiated by RNAPIIo-bound CSB, which recruits CSA through a newly identified CSA-interaction motif (CIM). Once recruited, CSA facilitates the association of UVSSA with stalled RNAPIIo. Importantly, we find that UVSSA is the key factor that recruits the TFIIH complex in a manner that is stimulated by CSB and CSA. Together these findings identify a sequential and highly cooperative assembly mechanism of TCR proteins and reveal the mechanism for TFIIH recruitment to DNA damage-stalled RNAPIIo to initiate repair.


Assuntos
Proteínas de Transporte/metabolismo , Dano ao DNA , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Polimerase II/metabolismo , Fator de Transcrição TFIIH/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Reparo do DNA , Humanos , Transcrição Genética , Raios Ultravioleta , Xenopus laevis
8.
Cell ; 181(2): 325-345.e28, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32302571

RESUMO

The mechanisms underlying ribonucleoprotein (RNP) granule assembly, including the basis for establishing and maintaining RNP granules with distinct composition, are unknown. One prominent type of RNP granule is the stress granule (SG), a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to stress. Here, we show that SGs assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein-RNA interaction network. The central node of this network is G3BP1, which functions as a molecular switch that triggers RNA-dependent LLPS in response to a rise in intracellular free RNA concentrations. Moreover, we show that interplay between three distinct intrinsically disordered regions (IDRs) in G3BP1 regulates its intrinsic propensity for LLPS, and this is fine-tuned by phosphorylation within the IDRs. Further regulation of SG assembly arises through positive or negative cooperativity by extrinsic G3BP1-binding factors that strengthen or weaken, respectively, the core SG network.


Assuntos
Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Ribonucleoproteínas/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Estruturas Citoplasmáticas/metabolismo , Células HEK293 , Humanos , Fosforilação , RNA/metabolismo
9.
Cell ; 181(2): 346-361.e17, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32302572

RESUMO

Stressed cells shut down translation, release mRNA molecules from polysomes, and form stress granules (SGs) via a network of interactions that involve G3BP. Here we focus on the mechanistic underpinnings of SG assembly. We show that, under non-stress conditions, G3BP adopts a compact auto-inhibited state stabilized by electrostatic intramolecular interactions between the intrinsically disordered acidic tracts and the positively charged arginine-rich region. Upon release from polysomes, unfolded mRNAs outcompete G3BP auto-inhibitory interactions, engendering a conformational transition that facilitates clustering of G3BP through protein-RNA interactions. Subsequent physical crosslinking of G3BP clusters drives RNA molecules into networked RNA/protein condensates. We show that G3BP condensates impede RNA entanglement and recruit additional client proteins that promote SG maturation or induce a liquid-to-solid transition that may underlie disease. We propose that condensation coupled to conformational rearrangements and heterotypic multivalent interactions may be a general principle underlying RNP granule assembly.


Assuntos
Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Células HeLa , Humanos , Conformação de Ácido Nucleico , Organelas/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Estresse Fisiológico/genética
10.
Cancer Res ; 80(8): 1735-1747, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32161100

RESUMO

Checkpoint kinase 1 (CHK1) is a key mediator of the DNA damage response that regulates cell-cycle progression, DNA damage repair, and DNA replication. Small-molecule CHK1 inhibitors sensitize cancer cells to genotoxic agents and have shown single-agent preclinical activity in cancers with high levels of replication stress. However, the underlying genetic determinants of CHK1 inhibitor sensitivity remain unclear. We used the developmental clinical drug SRA737 in an unbiased large-scale siRNA screen to identify novel mediators of CHK1 inhibitor sensitivity and uncover potential combination therapies and biomarkers for patient selection. We identified subunits of the B-family of DNA polymerases (POLA1, POLE, and POLE2) whose silencing sensitized the human A549 non-small cell lung cancer (NSCLC) and SW620 colorectal cancer cell lines to SRA737. B-family polymerases were validated using multiple siRNAs in a panel of NSCLC and colorectal cancer cell lines. Replication stress, DNA damage, and apoptosis were increased in human cancer cells following depletion of the B-family DNA polymerases combined with SRA737 treatment. Moreover, pharmacologic blockade of B-family DNA polymerases using aphidicolin or CD437 combined with CHK1 inhibitors led to synergistic inhibition of cancer cell proliferation. Furthermore, low levels of POLA1, POLE, and POLE2 protein expression in NSCLC and colorectal cancer cells correlated with single-agent CHK1 inhibitor sensitivity and may constitute biomarkers of this phenotype. These findings provide a potential basis for combining CHK1 and B-family polymerase inhibitors in cancer therapy. SIGNIFICANCE: These findings demonstrate how the therapeutic benefit of CHK1 inhibitors may potentially be enhanced and could have implications for patient selection and future development of new combination therapies.


Assuntos
Afidicolina/farmacologia , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Retinoides/farmacologia , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Dano ao DNA , DNA Polimerase I/antagonistas & inibidores , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , DNA Polimerase II/antagonistas & inibidores , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , DNA Polimerase beta , Drogas em Investigação/farmacologia , Inibidores Enzimáticos/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Interferente Pequeno/análise , RNA Interferente Pequeno/genética
11.
J Med Chem ; 63(7): 3508-3521, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32196342

RESUMO

We previously reported a first set of hybrid topoisomerase II (topoII) poisons whose chemical core merges key pharmacophoric elements of etoposide and merbarone, which are two well-known topoII blockers. Here, we report on the expansion of this hybrid molecular scaffold and present 16 more hybrid derivatives that have been designed, synthesized, and characterized for their ability to block topoII and for their overall drug-like profile. Some of these compounds act as topoII poison and exhibit good solubility, metabolic (microsomal) stability, and promising cytotoxicity in three cancer cell lines (DU145, HeLa, A549). Compound 3f (ARN24139) is the most promising drug-like candidate, with a good pharmacokinetics profile in vivo. Our results indicate that this hybrid new chemical class of topoII poisons deserves further exploration and that 3f is a favorable lead candidate as a topoII poison, meriting future studies to test its efficacy in in vivo tumor models.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Inibidores da Topoisomerase II/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/metabolismo , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/metabolismo , Inibidores da Topoisomerase II/farmacocinética
12.
Life Sci ; 248: 117467, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32105706

RESUMO

BACKGROUND: NQO1 protein acts as a cellular protective system, on account of its role as a quinone reductase and redox regulator. Nonetheless, new NQO1 roles are emerging-including its regulation of the cellular proliferation of many tumor cells-and this enzyme has been found to relate to the incidence of various diseases, including chronic myeloid leukemia. However, the mechanisms through which NQO1 influences leukemia progression remain unclear. MARTIAL AND METHODS: The current study looks to name NQO1 as a novel molecular target that modulates DNA synthesis and chronic myeloid leukemia growth. RESULTS AND CONCLUSION: Our results indicate that the frequency of the T allele of NQO1 polymorphism in chronic myeloid leukemia patients is higher than that among healthy East Asian individuals (0.492 vs. 0.419) and much higher than the average level of the general population (0.492 vs. 0.289) (1000 Genomes). Functionally, NQO1 knockdown increases the protein expression of the TOP2A and MCM complex, and consequently promotes DNA synthesis and K562 cell growth. NQO1 knockdown also promotes tumorigenesis in a xenograft model. NQO1 overexpression, on the other hand, was found to have the opposite effects. SIGNIFICANCE: Our results show that NQO1 downregulation promotes K562 cellular proliferation via the elevation of DNA synthesis.


Assuntos
DNA de Neoplasias/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucócitos/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , Adulto , Alelos , Animais , Grupo com Ancestrais do Continente Asiático , Linhagem Celular Tumoral , Proliferação de Células , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , DNA de Neoplasias/biossíntese , Feminino , Xenoenxertos , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/etnologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucócitos/patologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , NAD(P)H Desidrogenase (Quinona)/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Polimorfismo Genético , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
13.
PLoS Genet ; 16(2): e1008572, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32012149

RESUMO

Cancer genomes with mutations in the exonuclease domain of Polymerase Epsilon (POLE) present with an extraordinarily high somatic mutation burden. In vitro studies have shown that distinct POLE mutants exhibit different polymerase activity. Yet, genome-wide mutation patterns and driver mutation formation arising from different POLE mutants remains unclear. Here, we curated somatic mutation calls from 7,345 colorectal cancer samples from published studies and publicly available databases. These include 44 POLE mutant samples including 9 with whole genome sequencing data available. The POLE mutant samples were categorized based on the specific POLE mutation present. Mutation spectrum, associations of somatic mutations with epigenomics features and co-occurrence with specific driver mutations were examined across different POLE mutants. We found that different POLE mutants exhibit distinct mutation spectrum with significantly higher relative frequency of C>T mutations in POLE V411L mutants. Our analysis showed that this increase frequency in C>T mutations is not dependent on DNA methylation and not associated with other genomic features and is thus specifically due to DNA sequence context alone. Notably, we found strong association of the TP53 R213* mutation specifically with POLE P286R mutants. This truncation mutation occurs within the TT[C>T]GA context. For C>T mutations, this sequence context is significantly more likely to be mutated in POLE P286R mutants compared with other POLE exonuclease domain mutants. This study refines our understanding of DNA polymerase fidelity and underscores genome-wide mutation spectrum and specific cancer driver mutation formation observed in POLE mutant cancers.


Assuntos
Carcinogênese/genética , Neoplasias Colorretais/genética , DNA Polimerase II/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Domínios Proteicos/genética , Proteína Supressora de Tumor p53/genética , Ilhas de CpG/genética , Citosina/metabolismo , Metilação de DNA/genética , Análise Mutacional de DNA/estatística & dados numéricos , DNA Polimerase II/genética , Bases de Dados Genéticas/estatística & dados numéricos , Conjuntos de Dados como Assunto , Epigênese Genética , Humanos , Mutação , Proteínas de Ligação a Poli-ADP-Ribose/genética , Sequenciamento Completo do Genoma/estatística & dados numéricos
14.
Mol Cell ; 78(1): 70-84.e6, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32017897

RESUMO

Post-transcriptional mechanisms regulate the stability and, hence, expression of coding and noncoding RNAs. Sequence-specific features within the 3' untranslated region (3' UTR) often direct mRNAs for decay. Here, we characterize a genome-wide RNA decay pathway that reduces the half-lives of mRNAs based on overall 3' UTR structure formed by base pairing. The decay pathway is independent of specific single-stranded sequences, as regulation is maintained in both the original and reverse complement orientation. Regulation can be compromised by reducing the overall structure by fusing the 3' UTR with an unstructured sequence. Mutating base-paired RNA regions can also compromise this structure-mediated regulation, which can be restored by re-introducing base-paired structures of different sequences. The decay pathway requires the RNA-binding protein UPF1 and its associated protein G3BP1. Depletion of either protein increased steady-state levels of mRNAs with highly structured 3' UTRs as well as highly structured circular RNAs. This structure-dependent mechanism therefore enables cells to selectively regulate coding and noncoding RNAs.


Assuntos
Regiões 3' não Traduzidas , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Transativadores/metabolismo , Pareamento de Bases , Linhagem Celular , Regulação da Expressão Gênica , Humanos , RNA Circular/química , RNA Circular/metabolismo
15.
Proc Natl Acad Sci U S A ; 117(8): 4053-4060, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32041867

RESUMO

Small molecules can affect many cellular processes. The disambiguation of these effects to identify the causative mechanisms of cell death is extremely challenging. This challenge impacts both clinical development and the interpretation of chemical genetic experiments. CX-5461 was developed as a selective RNA polymerase I inhibitor, but recent evidence suggests that it may cause DNA damage and induce G-quadraplex formation. Here we use three complimentary data mining modalities alongside biochemical and cell biological assays to show that CX-5461 exerts its primary cytotoxic activity through topoisomerase II poisoning. We then show that acquired resistance to CX-5461 in previously sensitive lymphoma cells confers collateral resistance to the topoisomerase II poison doxorubicin. Doxorubicin is already a frontline chemotherapy in a variety of hematopoietic malignancies, and CX-5461 is being tested in relapse/refractory hematopoietic tumors. Our data suggest that the mechanism of cell death induced by CX-5461 is critical for rational clinical development in these patients. Moreover, CX-5461 usage as a specific chemical genetic probe of RNA polymerase I function is challenging to interpret. Our multimodal data-driven approach is a useful way to detangle the intended and unintended mechanisms of drug action across diverse essential cellular processes.


Assuntos
Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Naftiridinas/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Linfoma , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Interferência de RNA , Sensibilidade e Especificidade
16.
Nucleic Acids Res ; 48(5): 2473-2485, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31970402

RESUMO

Cockayne Syndrome (CS) is a rare neurodegenerative disease characterized by short stature, accelerated aging and short lifespan. Mutations in two human genes, ERCC8/CSA and ERCC6/CSB, are causative for CS and their protein products, CSA and CSB, while structurally unrelated, play roles in DNA repair and other aspects of DNA metabolism in human cells. Many clinical and molecular features of CS remain poorly understood, and it was observed that CSA and CSB regulate transcription of ribosomal DNA (rDNA) genes and ribosome biogenesis. Here, we investigate the dysregulation of rRNA synthesis in CS. We report that Nucleolin (Ncl), a nucleolar protein that regulates rRNA synthesis and ribosome biogenesis, interacts with CSA and CSB. In addition, CSA induces ubiquitination of Ncl, enhances binding of CSB to Ncl, and CSA and CSB both stimulate the binding of Ncl to rDNA and subsequent rRNA synthesis. CSB and CSA also increase RNA Polymerase I loading to the coding region of the rDNA and this is Ncl dependent. These findings suggest that CSA and CSB are positive regulators of rRNA synthesis via Ncl regulation. Most CS patients carry mutations in CSA and CSB and present with similar clinical features, thus our findings provide novel insights into disease mechanism.


Assuntos
Síndrome de Cockayne/genética , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Regulação da Expressão Gênica , Fosfoproteínas/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Ribossômico/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/metabolismo , Transcrição Genética , Linhagem Celular , DNA Ribossômico/genética , Humanos , Modelos Biológicos , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo
17.
PLoS Pathog ; 16(1): e1008250, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31905230

RESUMO

Viral infections impose major stress on the host cell. In response, stress pathways can rapidly deploy defence mechanisms by shutting off the protein synthesis machinery and triggering the accumulation of mRNAs into stress granules to limit the use of energy and nutrients. Because this threatens viral gene expression, viruses need to evade these pathways to propagate. Human norovirus is responsible for gastroenteritis outbreaks worldwide. Here we examined how norovirus interacts with the eIF2α signaling axis controlling translation and stress granules. While norovirus infection represses host cell translation, our mechanistic analyses revealed that eIF2α signaling mediated by the stress kinase GCN2 is uncoupled from translational stalling. Moreover, infection results in a redistribution of the RNA-binding protein G3BP1 to replication complexes and remodelling of its interacting partners, allowing the avoidance from canonical stress granules. These results define novel strategies by which norovirus undergo efficient replication whilst avoiding the host stress response and manipulating the G3BP1 interactome.


Assuntos
Infecções por Caliciviridae/virologia , DNA Helicases/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Norovirus/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Biossíntese de Proteínas , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Animais , Infecções por Caliciviridae/genética , Linhagem Celular , Grânulos Citoplasmáticos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células RAW 264.7 , RNA/metabolismo , Transdução de Sinais , Replicação Viral
18.
Nat Cell Biol ; 22(2): 175-186, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932739

RESUMO

Mouse embryonic stem cells (ESCs) sporadically transit into an early embryonic-like state characterized by the expression of 2-cell (2C) stage-restricted transcripts. Here, we identify a maternal factor-negative elongation factor A (NELFA)-whose heterogeneous expression in mouse ESCs is coupled to 2C gene upregulation and expanded developmental potential in vivo. We show that NELFA partners with Top2a in an interaction specific to the 2C-like state, and that it drives the expression of Dux-a key 2C regulator. Accordingly, loss of NELFA and/or Top2a suppressed Dux activation. Further characterization of 2C-like cells uncovered reduced glycolytic activity; remarkably, mere chemical suppression of glycolysis was sufficient to promote a 2C-like fate, obviating the need for genetic manipulation. Global chromatin state analysis on NELFA-induced cells revealed decommissioning of ESC-specific enhancers, suggesting ESC-state impediments to 2C reversion. Our study positions NELFA as one of the earliest drivers of the 2C-like state and illuminates factors and processes that govern this transition.


Assuntos
Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Murinas/metabolismo , Fatores de Transcrição/genética , Animais , Diferenciação Celular , Cromatina/química , Cromatina/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Embrião de Mamíferos , Feminino , Glicólise/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Padrões de Herança , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Família Multigênica , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo
19.
Nat Commun ; 11(1): 24, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911655

RESUMO

The spindle assembly checkpoint (SAC) prevents premature chromosome segregation by inactivating the anaphase promoting complex/cyclosome (APC/C) until all chromosomes are properly attached to mitotic spindles. Here we identify a role for Cullin-RING ubiquitin ligase complex 4 (CRL4), known for modulating DNA replication, as a crucial mitotic regulator that triggers the termination of the SAC and enables chromosome segregation. CRL4 is recruited to chromatin by the replication origin binding protein RepID/DCAF14/PHIP. During mitosis, CRL4 dissociates from RepID and replaces it with RB Binding Protein 7 (RBBP7), which ubiquitinates the SAC mediator BUB3 to enable mitotic exit. During interphase, BUB3 is protected from CRL4-mediated degradation by associating with promyelocytic leukemia (PML) nuclear bodies, ensuring its availability upon mitotic onset. Deficiencies in RepID, CRL4 or RBBP7 delay mitotic exit, increase genomic instability and enhance sensitivity to paclitaxel, a microtubule stabilizer and anti-tumor drug.


Assuntos
Anáfase , Proteínas de Ciclo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metáfase , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitose , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Ligação Proteica , Proteólise , Proteína 7 de Ligação ao Retinoblastoma/genética , Proteína 7 de Ligação ao Retinoblastoma/metabolismo , Fuso Acromático/metabolismo , Ubiquitina-Proteína Ligases/genética
20.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31941779

RESUMO

Stress granules (SGs) are formed in the cytoplasm under environmental stress, including viral infection. Human enterovirus D68 (EV-D68) is a highly pathogenic virus which can cause serious respiratory and neurological diseases. At present, there is no effective drug or vaccine against EV-D68 infection, and the relationship between EV-D68 infection and SGs is poorly understood. This study revealed the biological function of SGs in EV-D68 infection. Our results suggest that EV-D68 infection induced the accumulation of SG marker proteins Ras GTPase-activated protein-binding protein 1 (G3BP1), T cell intracellular antigen 1 (TIA1), and human antigen R (HUR) in the cytoplasm of infected host cells during early infection but inhibited their accumulation during the late stage. Simultaneously, we revealed that EV-D68 infection induces HUR, TIA1, and G3BP1 colocalization, which marks the formation of typical SGs dependent on protein kinase R (PKR) and eIF2α phosphorylation. In addition, we found that TIA1, HUR, and G3BP1 were capable of targeting the 3' untranslated regions (UTRs) of EV-D68 RNA to inhibit viral replication. However, the formation of SGs in response to arsenite (Ars) gradually decreased as the infection progressed, and G3BP1 was cleaved in the late stage as a strategy to antagonize SGs. Our findings have important implications in understanding the mechanism of interaction between EV-D68 and the host while providing a potential target for the development of antiviral drugs.IMPORTANCE EV-D68 is a serious threat to human health, and there are currently no effective treatments or vaccines. SGs play an important role in cellular innate immunity as a target with antiviral effects. This manuscript describes the formation of SGs induced by EV-D68 early infection but inhibited during the late stage of infection. Moreover, TIA1, HUR, and G3BP1 can chelate a specific site of the 3' UTR of EV-D68 to inhibit viral replication, and this interaction is sequence and complex dependent. However, this inhibition can be antagonized by overexpression of the minireplicon. These findings increase our understanding of EV-D68 infection and may help identify new antiviral targets that can inhibit viral replication and limit the pathogenesis of EV-D68.


Assuntos
Regiões 3' não Traduzidas , Grânulos Citoplasmáticos/virologia , Enterovirus Humano D/genética , Replicação Viral , Células A549 , Linhagem Celular Tumoral , Citoplasma/metabolismo , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Enterovirus Humano D/fisiologia , Células HEK293 , Células HeLa , Humanos , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA Interferente Pequeno/metabolismo , RNA Viral/metabolismo , Antígeno-1 Intracelular de Células T/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA