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1.
Anticancer Res ; 39(11): 6087-6095, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31704836

RESUMO

BACKGROUND: RAS GTPase-activating protein-binding protein (G3BP1) is an RNA-binding protein that is essential for assembling stress granules. Many functions related to the survival and progression of cancer have been reported. The current study aimed to investigate the role of G3BP1 in radio-sensitisation of cancer cells. MATERIALS AND METHODS: Radiation sensitivity and chemosensitivity were analysed in A549 and H460 cells transfected with G3BP1 siRNAs, and N-acetyl-L-cysteine (NAC) was used to elucidate the involvement of reactive oxygen species (ROS). RESULTS: G3BP1 depletion sensitised lung cancer cell lines to radiation, and the effect was related to ROS. G3BP1 depletion impaired the intracellular ROS scavenging system and NAC abolished the radiation-sensitive phenotypes caused by G3BP1 depletion. CONCLUSION: The study suggested G3BP1 as a promising target for radio- and chemosensitisation of lung cancer.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/radioterapia , Dano ao DNA/efeitos da radiação , DNA Helicases/antagonistas & inibidores , Neoplasias Pulmonares/radioterapia , Estresse Oxidativo/efeitos da radiação , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , RNA Helicases/antagonistas & inibidores , Proteínas com Motivo de Reconhecimento de RNA/antagonistas & inibidores , Tolerância a Radiação/efeitos dos fármacos , Antibióticos Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células , DNA Helicases/genética , DNA Helicases/metabolismo , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais Cultivadas
2.
Nucleic Acids Res ; 47(16): 8548-8562, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31276581

RESUMO

Cockayne syndrome is an accelerated aging disorder, caused by mutations in the CSA or CSB genes. In CSB-deficient cells, poly (ADP ribose) polymerase (PARP) is persistently activated by unrepaired DNA damage and consumes and depletes cellular nicotinamide adenine dinucleotide, which leads to mitochondrial dysfunction. Here, the distribution of poly (ADP ribose) (PAR) was determined in CSB-deficient cells using ADPr-ChAP (ADP ribose-chromatin affinity purification), and the results show striking enrichment of PAR at transcription start sites, depletion of heterochromatin and downregulation of H3K9me3-specific methyltransferases SUV39H1 and SETDB1. Induced-expression of SETDB1 in CSB-deficient cells downregulated PAR and normalized mitochondrial function. The results suggest that defects in CSB are strongly associated with loss of heterochromatin, downregulation of SETDB1, increased PAR in highly-transcribed regions, and mitochondrial dysfunction.


Assuntos
Senescência Celular/genética , Síndrome de Cockayne/genética , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Histonas/genética , Mitocôndrias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Metiltransferases de Proteína/genética , Fatores de Transcrição/genética , Linhagem Celular Transformada , Cromatina/química , Cromatina/metabolismo , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patologia , DNA/genética , DNA/metabolismo , Dano ao DNA , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Histonas/metabolismo , Humanos , Metiltransferases/genética , Metiltransferases/metabolismo , Mitocôndrias/patologia , Mutação , NAD/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Metiltransferases de Proteína/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Genética
3.
Oncol Rep ; 42(4): 1467-1474, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31322269

RESUMO

With the increasing use of poly(ADP­ribose) polymerase (PARP) inhibitors in cancer therapy, understanding their resistance is an urgent research quest. Additionally, CHFR is an E3 ubiquitin ligase, recruited to double­strand breaks (DSBs) by PAR. Furthermore, ALC1 is a new oncogene involved in the invasion and metastasis of breast cancer. Moreover, PARylated PARP1 activates ALC1 at sites of DNA damage, yet the underlying mechanism remains unclear. Mass spectrometric analysis, western blot analysis and immunoprecipitation were performed to confirm the interaction between CHFR and ALC1 in the physiological condition. Deletion mutants of CHFR and ALC1 were generated to map the interaction domain. PARP1/2 inhibitors were added to identify the ubiquitination of ALC1 by CHFR. ALC1 half­life was examined to compare the expression of ALC1 protein in the presence and absence of PARP1/2 inhibitors. The results revealed that the transcriptional level of ALC1 was not upregulated in breast cancer tissues. CHFR interacted with ALC1. The PBZ domain of CHFR, the PMD domain and the MACRO domain of ALC1 domain are the necessary regions for the interaction depending on PAR. Ubiquitination of ALC1 by CHFR was dependent on PARylation and resulted in the degradation of PARylated ALC1. PARP1/2 inhibitors decreased the ubiquitination of PAR­dependent ALC1, and the expression of ALC1 was upregulated by PARP1/2 inhibitors. Ubiquitination mediated by CHFR resulted in the degradation of ALC1. In conclusion, PARP1/2 inhibitors decrease the ubiquitination of ALC1 leading to the accumulation of ALC1, which affects the therapeutic effects of DNA damage response drugs in breast cancer treatment.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Neoplasias/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Células MCF-7 , Poli(ADP-Ribose) Polimerase-1/metabolismo , Transcrição Genética , Ubiquitinação/efeitos dos fármacos
4.
PLoS Pathog ; 15(6): e1007842, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31199850

RESUMO

G3BP-1 and -2 (hereafter referred to as G3BP) are multifunctional RNA-binding proteins involved in stress granule (SG) assembly. Viruses from diverse families target G3BP for recruitment to replication or transcription complexes in order to block SG assembly but also to acquire pro-viral effects via other unknown functions of G3BP. The Old World alphaviruses, including Semliki Forest virus (SFV) and chikungunya virus (CHIKV) recruit G3BP into viral replication complexes, via an interaction between FGDF motifs in the C-terminus of the viral non-structural protein 3 (nsP3) and the NTF2-like domain of G3BP. To study potential proviral roles of G3BP, we used human osteosarcoma (U2OS) cell lines lacking endogenous G3BP generated using CRISPR-Cas9 and reconstituted with a panel of G3BP1 mutants and truncation variants. While SFV replicated with varying efficiency in all cell lines, CHIKV could only replicate in cells expressing G3BP1 variants containing both the NTF2-like and the RGG domains. The ability of SFV to replicate in the absence of G3BP allowed us to study effects of different domains of the protein. We used immunoprecipitation to demonstrate that that both NTF2-like and RGG domains are necessary for the formation a complex between nsP3, G3BP1 and the 40S ribosomal subunit. Electron microscopy of SFV-infected cells revealed that formation of nsP3:G3BP1 complexes via the NTF2-like domain was necessary for clustering of cytopathic vacuoles (CPVs) and that the presence of the RGG domain was necessary for accumulation of electron dense material containing G3BP1 and nsP3 surrounding the CPV clusters. Clustered CPVs also exhibited localised high levels of translation of viral mRNAs as detected by ribopuromycylation staining. These data confirm that G3BP is a ribosomal binding protein and reveal that alphaviral nsP3 uses G3BP to concentrate viral replication complexes and to recruit the translation initiation machinery, promoting the efficient translation of viral mRNAs.


Assuntos
Proteínas de Transporte/metabolismo , Febre de Chikungunya/metabolismo , Vírus Chikungunya/fisiologia , DNA Helicases/metabolismo , Iniciação Traducional da Cadeia Peptídica , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Vírus da Floresta de Semliki/fisiologia , Replicação Viral , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Febre de Chikungunya/genética , Febre de Chikungunya/patologia , Cricetinae , DNA Helicases/genética , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Domínios Proteicos , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo
5.
BMC Cancer ; 19(1): 604, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31216997

RESUMO

BACKGROUND: The prognosis of bladder urothelial carcinoma (BLCA) varies greatly among patients, and conventional pathological predictors are generally inadequate and often inaccurate to predict the heterogeneous behavior of BLCA. This study aims to investigate the prognostic value and function of TOP2A in BLCA. METHODS: TOP2A expression level was examined by RNA-sequencing, quantitative real time polymerase chain reaction and immunohistochemistry from 10, 40 and 209 BLCA samples, respectively. Public databases were analyzed for validation. Cell proliferation, migration, invasion assays were performed to explore potential functions of TOP2A in BLCA. Flow cytometry was performed for cell cycle and apoptosis analysis. Univariable and multivariable Cox regression models were performed to identify independent risk factors for the prognosis of BLCA. RESULTS: We found TOP2A was significantly upregulated in BLCA samples, especially for high-grade and advanced stage tumors, compared with matched normal epithelial tissue. Univariable COX regression analysis revealed high TOP2A expression was significantly associated with poorer cancer-specific, progression-free and recurrence-free survival, but not independently of clinical characteristics in the multivariable models. Knockdown of TOP2A remarkably inhibited the proliferation of BLCA cells and non-cancerous urothelial cells. Furthermore, migration and invasion capacity of BLCA cells were strongly suppressed after TOP2A knockdown. Moreover, flow cytometry suggested TOP2A had anti-apoptotic function, and knockdown of TOP2A could induce resistance to doxorubicin in J82 cells. CONCLUSIONS: In our study, TOP2A was overexpressed in BLCA and could serve as a prognostic biomarker for BLCA. Moreover, TOP2A is functionally important for the proliferation, invasion and survival of BLCA cells.


Assuntos
Carcinoma/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Idoso , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma/genética , Carcinoma/patologia , Linhagem Celular Tumoral , Proliferação de Células , DNA Topoisomerases Tipo II/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Proteínas de Ligação a Poli-ADP-Ribose/genética , Prognóstico , Modelos de Riscos Proporcionais , Análise de Sequência de RNA , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia
6.
J Biosci ; 44(2)2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31180040

RESUMO

Human Y-box binding protein-1 (YBX1) is a member of highly conserved cold-shock domain protein family, which is involved in transcriptional as well as translational regulation of many genes. Nuclear localization of YBX1 has been observed in various cancer types and it's overexpression has been linked to adverse clinical outcome and poor therapy response, but no diagnostic or therapeutic correlation has been established so far. This study aimed to identify differentially expressed novel genes among the interactors of YBX1 in different cancer types. Analysis of RNA-Seq data for colorectal, lung, prostate and stomach adenocarcinoma identified 39 unique genes, which are differentially expressed in the four adenocarcinoma types. Gene-enrichment analysis for the differentially expressed genes from individual adenocarcinoma with focus on unique genes resulted in a total of 57 gene sets specific to each adenocarcinoma. Gene ontology for commonly expressed genes suggested the pathways and possible mechanisms through which they affect each adenocarcinoma type considered in the study. Gene regulatory network constructed for the common genes and network topology was analyzed for the central nodes. Here 12 genes were found to play important roles in the network formation; among them, two genes FOXM1 and TOP2A were found to be in central network formation, which makes them a common target for therapeutics. Furthermore, five common differentially expressed genes in all adenocarcinomas were also identified.


Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias da Próstata/genética , Neoplasias Gástricas/genética , Proteína 1 de Ligação a Y-Box/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Biologia Computacional/métodos , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Feminino , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Perfilação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Anotação de Sequência Molecular , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Mapeamento de Interação de Proteínas , Transdução de Sinais , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Proteína 1 de Ligação a Y-Box/metabolismo
7.
Mol Cell Biochem ; 459(1-2): 189-204, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31172368

RESUMO

G3BPs are members of an RNA-binding protein family and their aberrant expression is common in various cancers and there is growing evidence that G3BPs possess antiviral activities and are targeted by various viruses. G3BPs have also been implicated in both stabilization and degradation of specific mRNAs as well as translational control of mRNA targets. G3BPs have been shown to control translation of interferon-stimulated genes (ISGs), implying that G3BPs are involved in the regulation of the interferon system in response to viral infections and/or cellular stress. The interferon induced transmembrane (IFITM1, IFITM2 and IFITM3) proteins are antiviral proteins, and are also involved in cancer progression and metastasis. Therefore, these genes were selected in the studies reported here as potential transcript targets of G3BPs. Furthermore, G3BPs are involved in the regulation of the MEK pathway which also impacts on the translation of ISGs. Therefore, the role of this pathway was also analysed in regulation of IFITM1-3 proteins. Overall, this research study suggests that G3BPs are essential for the accumulation of IFITM1-3 proteins and intersect twice in the regulation of IFITM1-3 expression, first through MEK pathway and then through an interaction with the 3'-UTRs of its target transcripts. However, it is still to be determined whether the two apparent functions are part of a single control mechanism or the two functions are mutually exclusive.


Assuntos
Antígenos de Diferenciação/biossíntese , Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/biossíntese , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Biossíntese de Proteínas , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA/biossíntese , Antígenos de Diferenciação/genética , Proteínas de Transporte/genética , DNA Helicases/genética , Humanos , Células MCF-7 , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas de Ligação a RNA/genética
8.
BMC Cancer ; 19(1): 429, 2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31072339

RESUMO

BACKGROUND: Despite rising incidence rates of colorectal malignancies, only a few prognostic tools have been implemented in proven clinical routine. Cell division and proliferation play a significant role in malignancies. In terms of colorectal cancer, the impact of proliferation associated proteins is controversially debated. The aim of our study was to examine the expression of topoisomerase II α and minichromosome maintenance protein 6 and to correlate these findings with the clinical data. METHODS: Tissue samples of 619 patients in total were stained using the antibodies Ki-S4 and Ki-MCM6 targeting topoisomerase II α as well as minichromosome maintenance protein 6. The median rate of proliferation was correlated with clinical and follow up data. RESULTS: The expression rate of minichromosome maintenance protein 6 is significantly higher than the proportion of topoisomerase II α in tumour cells (p < 0.001). A high expression of both proteins coincides with a beneficial outcome for the patient, indicating a favourable prognostic marker (p < 0.001 and p = 0.008). CONCLUSIONS: We have demonstrated that high expression rates of proliferative markers is linked to a beneficial patient outcome. According to the general opinion, a high expression rate correlates with a poor patient outcome. In this study, we were able to refute this assertion.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , DNA Topoisomerases Tipo II/metabolismo , Componente 6 do Complexo de Manutenção de Minicromossomo/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Idoso , Proliferação de Células , Colo/patologia , Colo/cirurgia , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/cirurgia , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Reto/patologia , Reto/cirurgia , Estudos Retrospectivos , Análise de Sobrevida
9.
Biochim Biophys Acta Gene Regul Mech ; 1862(6): 657-669, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31075539

RESUMO

Variation in Disrupted-in-Schizophrenia 1 (DISC1) increases the risk for neurodegenerative diseases, schizophrenia, and other mental disorders. However, the functions of DISC1 associated with the development of these diseases remain unclear. DISC1 has been reported to inhibit Akt/mTORC1 signaling, a major regulator of translation, and recent studies indicate that DISC1 could exert a direct role in translational regulation. Here, we present evidence of a novel role of DISC1 in the maintenance of protein synthesis during oxidative stress. In order to investigate DISC1 function independently of Akt/mTORC1, we used Tsc2-/- cells, where mTORC1 activation is independent of Akt. DISC1 knockdown enhanced inhibition of protein synthesis in cells treated with sodium arsenite (SA), an oxidative agent used for studying stress granules (SGs) dynamics and translational control. N-acetyl-cysteine inhibited the effect of DISC1, suggesting that DISC1 affects translation in response to oxidative stress. DISC1 decreased SGs number in SA-treated cells, but resided outside SGs and maintained protein synthesis independently of a proper SG nucleation. DISC1-dependent stimulation of translation in SA-treated cells was supported by its interaction with eIF3h, a component of the canonical translation initiation machinery. Consistent with a role in the homeostatic maintenance of translation, DISC1 knockdown or overexpression decreased cell viability after SA exposure. Our data suggest that DISC1 is a relevant component of the cellular response to stress, maintaining certain levels of translation and preserving cell integrity. This novel function of DISC1 might be involved in its association with pathologies affecting tissues frequently exposed to oxidative stress.


Assuntos
Arsenitos/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Compostos de Sódio/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Proteínas do Tecido Nervoso/genética , Proteína Oncogênica v-akt , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Transcriptoma , Proteína 2 do Complexo Esclerose Tuberosa/genética
10.
Int J Mol Sci ; 20(5)2019 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-30871006

RESUMO

Type II topoisomerase enzymes are essential for resolving DNA topology problems arising through various aspects of DNA metabolism. In vertebrates two isoforms are present, one of which (TOP2A) accumulates on chromatin during mitosis. Moreover, TOP2A targets the mitotic centromere during prophase, persisting there until anaphase onset. It is the catalytically-dispensable C-terminal domain of TOP2 that is crucial in determining this isoform-specific behaviour. In this study we show that, in addition to the recently identified chromatin tether domain, several other features of the alpha-C-Terminal Domain (CTD). influence the mitotic localisation of TOP2A. Lysine 1240 is a major SUMOylation target in cycling human cells and the efficiency of this modification appears to be influenced by T1244 and S1247 phosphorylation. Replacement of K1240 by arginine results in fewer cells displaying centromeric TOP2A accumulation during prometaphase-metaphase. The same phenotype is displayed by cells expressing TOP2A in which either of the mitotic phosphorylation sites S1213 or S1247 has been substituted by alanine. Conversely, constitutive modification of TOP2A by fusion to SUMO2 exerts the opposite effect. FRAP analysis of protein mobility indicates that post-translational modification of TOP2A can influence the enzyme's residence time on mitotic chromatin, as well as its subcellular localisation.


Assuntos
Anáfase/fisiologia , Cromatina/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Metáfase/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Linhagem Celular , Centrômero/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Fosforilação/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação/fisiologia
11.
Oncol Rep ; 41(4): 2440-2452, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30816525

RESUMO

Adrenocortical carcinoma (ACC) is a rare disease associated with a poor prognosis. Furthermore, the underlying molecular mechanism of carcinogenesis is poorly understood, and prognostic prediction of ACC has low accuracy. In the present study, a bioinformatics approach was used to investigate the molecular mechanisms and prognosis of ACC. Samples of adrenal tumors were collected from patients undergoing adrenalectomy at the Department of Urology, the First Hospital of China Medical University. The analyzed gene datasets were downloaded from the Gene Expression Omnibus and The Cancer Genome Atlas (TCGA) database. Following this, the differentially expressed genes (DEGs) were included in Gene Ontology enrichment, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, protein­protein interaction network and survival analyses. MTT colorimetric assays, colony formation assays and 5­ethynyl­20­deoxyuridine incorporation assays were also conducted to evaluate ACC cell proliferation. The identified DEGs included 20 downregulated genes and 51 upregulated genes, which were highly associated with the cell cycle, organelle fission, chromosome segregation, cell division and spindle stability. The top 14 hub genes were subsequently confirmed by reverse transcription­quantitative polymerase chain reaction in ACC and adrenocortical adenoma samples. It was identified that the nuclear division cycle 80, cyclin B2 and topoisomerase 2­α may serve important roles in adrenocortical tumor development. Furthermore, these three genes predicted overall survival and recurrence­free survival in patients with ACC from the TCGA cohort. The findings identified three novel genes that have important roles in carcinogenesis and in the prognostic prediction of ACC.


Assuntos
Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/patologia , Ciclina B2/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Recidiva Local de Neoplasia/diagnóstico , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Córtex Suprarrenal/patologia , Córtex Suprarrenal/cirurgia , Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/mortalidade , Adrenalectomia , Carcinoma Adrenocortical/genética , Carcinoma Adrenocortical/mortalidade , Carcinogênese/genética , Carcinogênese/patologia , Proliferação de Células/genética , Biologia Computacional , Ciclina B2/genética , DNA Topoisomerases Tipo II/genética , Bases de Dados Genéticas/estatística & dados numéricos , Conjuntos de Dados como Assunto , Intervalo Livre de Doença , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Proteínas Nucleares/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Prognóstico , Mapas de Interação de Proteínas/genética , RNA Interferente Pequeno/metabolismo , Regulação para Cima
12.
Comput Biol Chem ; 79: 73-82, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30731361

RESUMO

Computational quantum chemical study and biological evaluation of a synthesized novel sulfur heterocyclic thiophene derivative containing 1,2,3-triazole and pyridine moieties namely BTPT [2-(1-benzyl-5-methyl-1H-1,2,3-triazol-4-yl)-6-methoxy-4-(thiophen-2-yl) pyridine] was presented in this study. The crystal structure was determined by SCXRD method. For the title compound BTPT, spectroscopic characterization like 1H NMR, 13C NMR, FTIR, UV-vis were carried out theoretically by computational DFT method and compared with experimental data. Druglikeness parameters of BTPT were found through in silico pharmacological ADMET properties estimation. The molecular docking investigation was performed with human topoisomerase IIα (PDB ID:1ZXM) targeting ATP binding site. In vitro cytotoxicity activity of BTPT/doxorubicin were examined by MTT assay procedure against three human cancer cell lines A549, PC-3, MDAMB-231 with IC50 values of 0.68/0.70, 1.03/0.77 and 0.88/0.98 µM, respectively. Our title compound BTPT reveals notable cytotoxicity against breast cancer cell (MDAMB-231), moderate activity with human lung cancer cell (A-549) and less inhibition with human prostate cancer cell (PC-3) compared to familiar cancer medicine doxorubicin. From the results, BTPT could be observed as a potential candidate for novel anticancer drug development process.


Assuntos
Antineoplásicos/farmacologia , Teoria da Densidade Funcional , Simulação de Acoplamento Molecular , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Inibidores da Topoisomerase II/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Humanos , Estrutura Molecular , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Piridinas/química , Piridinas/farmacologia , Relação Estrutura-Atividade , Enxofre/química , Enxofre/farmacologia , Tiofenos/química , Tiofenos/farmacologia , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/química , Triazóis/química , Triazóis/farmacologia
13.
Nucleic Acids Res ; 47(8): 4011-4025, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30715484

RESUMO

Transcription-coupled nucleotide excision repair (TC-NER) is a dedicated DNA repair pathway that removes transcription-blocking DNA lesions (TBLs). TC-NER is initiated by the recognition of lesion-stalled RNA Polymerase II by the joint action of the TC-NER factors Cockayne Syndrome protein A (CSA), Cockayne Syndrome protein B (CSB) and UV-Stimulated Scaffold Protein A (UVSSA). However, the exact recruitment mechanism of these factors toward TBLs remains elusive. Here, we study the recruitment mechanism of UVSSA using live-cell imaging and show that UVSSA accumulates at TBLs independent of CSA and CSB. Furthermore, using UVSSA deletion mutants, we could separate the CSA interaction function of UVSSA from its DNA damage recruitment activity, which is mediated by the UVSSA VHS and DUF2043 domains, respectively. Quantitative interaction proteomics showed that the Spt16 subunit of the histone chaperone FACT interacts with UVSSA, which is mediated by the DUF2043 domain. Spt16 is recruited to TBLs, independently of UVSSA, to stimulate UVSSA recruitment and TC-NER-mediated repair. Spt16 specifically affects UVSSA, as Spt16 depletion did not affect CSB recruitment, highlighting that different chromatin-modulating factors regulate different reaction steps of the highly orchestrated TC-NER pathway.


Assuntos
Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , DNA/genética , Proteínas de Grupo de Alta Mobilidade/genética , RNA Polimerase II/genética , Fatores de Transcrição/genética , Transcrição Genética , Fatores de Elongação da Transcrição/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Cromatina/metabolismo , Cromatina/ultraestrutura , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Helicases/genética , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Imagem Óptica , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Ligação Proteica , Domínios Proteicos , Transporte Proteico , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/metabolismo
14.
J Biol Chem ; 294(16): 6430-6438, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30804210

RESUMO

RIG-I senses viral RNA in the cytosol and initiates host innate immune response by triggering the production of type 1 interferon. A recent RNAi knockdown screen yielded close to hundred host genes whose products affected viral RNA-induced IFN-ß production and highlighted the complexity of the antiviral response. The stress granule protein G3BP1, known to arrest mRNA translation, was identified as a regulator of RIG-I-induced IFN-ß production. How G3BP1 functions in RIG-I signaling is not known, however. Here, we overexpress G3BP1 with RIG-I in HEK293T cells and found that G3BP1 significantly enhances RIG-I-induced ifn-b mRNA synthesis. More importantly, we demonstrate that G3BP1 binds RIG-I and that this interaction involves the C-terminal RGG domain of G3BP1. Confocal microscopy studies also show G3BP1 co-localization with RIG-I and with infecting vesicular stomatitis virus in Cos-7 cells. Interestingly, immunoprecipitation studies using biotin-labeled viral dsRNA or poly(I·C) and cell lysate-derived or in vitro translated G3BP1 indicated that G3BP1 could directly bind these substrates and again via its RGG domain. Computational modeling further revealed a juxtaposed interaction between G3BP1 RGG and RIG-I RNA-binding domains. Together, our data reveal G3BP1 as a critical component of RIG-I signaling and possibly acting as a co-sensor to promote RIG-I recognition of pathogenic RNA.


Assuntos
Proteína DEAD-box 58 , DNA Helicases , Interferon beta , Modelos Moleculares , Proteínas de Ligação a Poli-ADP-Ribose , Biossíntese de Proteínas , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , RNA de Cadeia Dupla , RNA Viral , Infecções por Rhabdoviridae , Vesiculovirus , Animais , Células COS , Cercopithecus aethiops , Proteína DEAD-box 58/química , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Células HEK293 , Humanos , Interferon beta/biossíntese , Interferon beta/genética , Camundongos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Ligação Proteica , Células RAW 264.7 , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Infecções por Rhabdoviridae/genética , Infecções por Rhabdoviridae/metabolismo , Transdução de Sinais/genética , Vesiculovirus/química , Vesiculovirus/genética , Vesiculovirus/metabolismo
15.
Biochim Biophys Acta Mol Cell Res ; 1866(3): 360-370, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30595162

RESUMO

Ras-GTPase-activating protein (SH3 domain)-binding proteins (G3BPs, also known as Rasputin) are a family of RNA binding proteins that regulate gene expression in response to environmental stresses by controlling mRNA stability and translation. G3BPs appear to facilitate this activity through their role in stress granules for which they are considered a core component, however, it should be noted that not all stress granules contain G3BPs and this appears to be contextual depending on the environmental stress and the cell type. Although the role of G3BPs in stress granules appears to be one of its major roles, data also strongly suggests that they interact with mRNAs outside of stress granules to regulate gene expression. G3BPs have been implicated in several diseases including cancer progression, invasion, and metastasis as well as virus survival. There is now a body of evidence that suggests targeting of G3BPs could be explored as a form of cancer therapeutic. This review discusses the important discoveries and advancements made in the field of G3BPs biology over the last two decades including their roles in RNA stability, translational control of cellular transcripts, stress granule formation, cancer progression and its interactions with viruses during infection. An emerging theme for G3BPs is their ability to regulate gene expression in response to environmental stimuli, disease progression and virus infection making it an intriguing target for disease therapies.


Assuntos
DNA Helicases/metabolismo , DNA Helicases/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/fisiologia , RNA Helicases/metabolismo , RNA Helicases/fisiologia , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/fisiologia , Estresse Fisiológico/fisiologia , Animais , Proteínas de Transporte/metabolismo , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/genética , Humanos , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais
16.
Phytomedicine ; 54: 109-119, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30668360

RESUMO

BACKGROUND: Phytochemical naphtho[1,2-b] furan-4,5­dione (NFD) presenting in Avicennia marina exert anti-cancer effects, but little is known regarding about DNA damage-mediated apoptosis in non-small-cell lung carcinoma (NSCLC). PURPOSE: To examine whether NFD-induced apoptosis of NSCLC cells is correlated with the induction of DNA damage, and to investigate its underlying mechanism. STUDY DESIGN: The anti-proliferative effects of NFD were assessed by MTS Assay Kit FACS assay, and in vivo nude mice xenograft assay. The DNA damage related proteins, the Bcl-2 family and pro-apoptotic factors were examined by immunofluorescence assay, q-PCR, and western blotting. The activity of NF-κB p65 in nuclear extracts was detected using a colorimetric DNA-binding ELISA assay. The inhibitory activity of topoisomerase II (TOPO II) was evaluated by molecular docking and TOPO II catalytic assay. RESULTS: NFD exerted selective cytotoxicity against NSCLC H1299, H1437 and A549 cells rather than normal lung-embryonated cells MRC-5. Remarkably, we found that NFD activated the hull marker and modulator of DNA damage repairs such as γ-H2AX, ATM, ATR, CHK1, and CHK2 probably caused by the accumulation of intracellular reactive oxygen species (ROS) and inhibition of TOPO II activity. Furthermore, the suppression of transcription factor NF-κB by NFD resulted in significantly decreased levels of pro-survival proteins including Bcl-2 family Bcl-2, Bcl-xL and Mcl-1 and the endogenous inhibitors of apoptosis XIAP and survivin in H1299 cells. Moreover, the nude mice xenograft assay further validated the suppression of H1299 growth by NFD, which is the first report for evaluating the anti-cancer effect of NFD in vivo. CONCLUSION: These findings provide a novel mechanism indicating the inhibition of TOPO II activity and NF-κB signaling by NFD, leading to DNA damage and apoptosis of NSCLC tumor cells.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Dano ao DNA/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , Furanos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Naftoquinonas/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Animais , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/química , Feminino , Furanos/química , Humanos , Neoplasias Pulmonares/genética , Camundongos Nus , Simulação de Acoplamento Molecular , NF-kappa B/metabolismo , Naftoquinonas/química , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/química , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Molecules ; 23(12)2018 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-30544723

RESUMO

Glioblastoma (GBM), the most common type of primary tumor in the central nervous system, is a very aggressive brain tumor with poor prognosis and a high recurrence rate. Increasing evidence suggests that human cytomegalovirus (HCMV) infection is related to GBM and leads to GBM cell growth and metastasis. MicroRNAs are important regulators in the growth and metastasis of glioblastoma. This study aimed to demonstrate the role of miR-144-3p in HCMV-positive glioblastoma. We found that, after HCMV infection, the expression of miR-144-3p decreased, whereas the expression of TOP2A increased. Bioinformatics analyses indicated that miR-144-3p directly targets the TOP2A 3'-UTR (Untranslated Region). We discovered that the overexpression of miR-144-3p downregulated the overexpression of TOP2A and inhibited the proliferation, clone formation, and invasion of HCMV-positive glioma in vitro. Taken together, these results show that miR-144-3p inhibited growth and promoted apoptosis in glioma cells by targeting TOP2A.


Assuntos
Neoplasias Encefálicas/patologia , Infecções por Citomegalovirus/genética , DNA Topoisomerases Tipo II/genética , Glioblastoma/patologia , MicroRNAs/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Regiões 3' não Traduzidas , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/virologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/patologia , DNA Topoisomerases Tipo II/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/virologia , Humanos , Masculino , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo
18.
Mol Cancer ; 17(1): 174, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30553276

RESUMO

BACKGROUND: Chromatin modification at mitosis is closely related to transcriptional reactivation in the subsequent cell cycle. We reasoned this process is deregulated by oncogenic signals, which would contribute to mitotic stress resistance in pancreatic cancer. Here, we show DMAP1/Bub3 complex mediates mitotic stress-induced cellular apoptosis, while this effect is counteracted by c-Src in pancreatic cancer cells. Our study aims to uncover an unidentified mechanism underlying the distinct response to mitotic stress between normal cells and pancreatic cancer cells. METHODS: The interaction between Bub3 and DMAP1 upon mitotic stress signaling was determined through molecular and cell biological methods. The inhibitory effect of c-Src on DMAP1/Bub3-mediated DNA methylation and gene transcription profile was investigated. The association between c-Src-mediated DMAP1 phosphorylation and paclitaxel activity in vivo and clinicopathologic characteristics were analyzed. RESULTS: Mitotic arrest induced p38-dependent phosphorylation of Bub3 at Ser211, which promotes DMAP1/Bub3 interaction. DMAP1/Bub3 complex is recruited by TAp73 to the promoter of anti-apoptotic gene BCL2L1, thus mediates the DNA methylation and represses gene transcription linked to cell apoptosis. Meanwhile, DMAP1 was highly phosphorylated at Tyr 246 by c-Src in pancreatic cancer cells, which impedes DMAP1/Bub3 interaction and the relevant cellular activites. Blocking DMAP1 pTyr-246 potentiates paclitaxel-inhibited tumor growth. Clinically, DMAP1 Tyr 246 phosphorylation correlates with c-Src activity in human pancreatic cancer specimens and poor prognosis in pancreatic cancer patients. CONCLUSIONS: Our findings reveal a regulatory role of Bub3 in DMAP1-mediated DNA methylation upon mitotic stress and provide the relevance of DMAP1 pTyr-246 to mitotic stress resistance during pancreatic cancer treatment.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Mitose/fisiologia , Neoplasias Pancreáticas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas Repressoras/antagonistas & inibidores , Quinases da Família src/metabolismo , Animais , Apoptose/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Metilação de DNA , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Nus , Mitose/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Transcrição Genética , Quinases da Família src/genética
19.
Int J Mol Sci ; 19(7)2018 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-30011940

RESUMO

DNA topoisomerase II (TOP2) activity involves a normally transient double-strand break intermediate in which the enzyme is coupled to DNA via a 5'-phosphotyrosyl bond. However, etoposide and other topoisomerase drugs poison the enzyme by stabilising this enzyme-bridged break, resulting in the accumulation of TOP2-DNA covalent complexes with cytotoxic consequences. The phosphotyrosyl diesterase TDP2 appears to be required for efficient repair of this unusual type of DNA damage and can remove 5'-tyrosine adducts from a double-stranded oligonucleotide substrate. Here, we adapt the trapped in agarose DNA immunostaining (TARDIS) assay to investigate the role of TDP2 in the removal of TOP2-DNA complexes in vitro and in cells. We report that TDP2 alone does not remove TOP2-DNA complexes from genomic DNA in vitro and that depletion of TDP2 in cells does not slow the removal of TOP2-DNA complexes. Thus, if TDP2 is involved in repairing TOP2 adducts, there must be one or more prior steps in which the protein-DNA complex is processed before TDP2 removes the remaining 5' tyrosine DNA adducts. While this is partly achieved through the degradation of TOP2 adducts by the proteasome, a proteasome-independent mechanism has also been described involving the SUMOylation of TOP2 by the ZATT E3 SUMO ligase. The TARDIS assay was also adapted to measure the effect of TDP2 knockdown on levels of SUMOylated TOP2-DNA complexes, which together with levels of double strand breaks were unaffected in K562 cells following etoposide exposure and proteasomal inhibition.


Assuntos
Reparo do DNA , DNA Topoisomerases Tipo II/metabolismo , DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Fatores de Transcrição/metabolismo , DNA/genética , Adutos de DNA/genética , Adutos de DNA/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Topoisomerases Tipo II/genética , Etoposídeo/farmacologia , Humanos , Células K562 , Proteínas Nucleares/genética , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/genética , Interferência de RNA , Sumoilação , Inibidores da Topoisomerase II/farmacologia , Fatores de Transcrição/genética
20.
DNA Repair (Amst) ; 68: 58-67, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29957539

RESUMO

The ATP-dependent chromatin remodeler CSB is implicated in a variety of different DNA repair mechanisms, including transcription-coupled nucleotide excision repair (TC-NER), base excision repair and DNA double strand break (DSB) repair. However, how CSB is regulated in these various repair processes is not well understood. Here we report that the first 30 amino acids of CSB along with two phosphorylation events on S10 and S158, previously reported to be required for CSB function in homologous recombination (HR)-mediated repair, are dispensable for repairing UV-induced DNA damage, suggesting that the regulation of CSB in these two types of repair are carried out by distinct mechanisms. In addition, we show that although the central ATPase domain of CSB is engaged in interactions with both the N- and C-terminal regions, these interactions are disrupted following UV-induced DNA damage. The UV-induced disengagement of the C-terminal region of CSB from the ATPase domain requires two conserved amino acids W1486 and L1488, which are thought to contribute to the hydrophobic core formation of the winged helix domain (WHD) at its C-terminus. Failure to undergo UV-induced dissociation of the C-terminal region of CSB from the ATPase domain is associated with impairment in its UV-induced chromatin association, its UV-induced post-translational modification as well as cell survival. Collectively, these findings suggest that UV-induced dissociation of CSB domain interactions is a necessary step in repairing UV-induced DNA damage and that the WHD of CSB plays a key role in this dissociation.


Assuntos
Dano ao DNA , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Domínios Proteicos , Raios Ultravioleta , Adenosina Trifosfatases , Linhagem Celular , Síndrome de Cockayne , DNA/metabolismo , DNA/efeitos da radiação , DNA Helicases/efeitos da radiação , Enzimas Reparadoras do DNA/efeitos da radiação , Humanos , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose/efeitos da radiação , Processamento de Proteína Pós-Traducional
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