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1.
Medicine (Baltimore) ; 98(30): e16534, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31348270

RESUMO

BACKGROUND: High-grade prostate cancer (PCa) has a poor prognosis, and up to 15% of patients worldwide experience lymph node invasion (LNI). To further improve the prediction lymph node invasion in prostate cancer, we adopted risk scores of the genes expression based on the nomogram in guidelines. METHODS: We analyzed clinical data from 320 PCa patients from the Cancer Genome Atlas database. Weighted gene coexpression network analysis was used to identify the genes that were significantly associated with LNI in PCa (n = 390). Analyses using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases were performed to identify the activated signaling pathways. Univariate and multivariate logistic regression analyses were performed to identify the independent risk factors for the presence of LNI. RESULTS: We found that patients with actual LNI and predicted LNI had the worst survival outcomes. The 7 most significant genes (CTNNAL1, ENSA, MAP6D1, MBD4, PRCC, SF3B2, TREML1) were selected for further analysis. Pathways in the cell cycle, DNA replication, oocyte meiosis, and 9 other pathways were dramatically activated during LNI in PCa. Multivariate analyses identified that the risk score (odds ratio [OR] = 1.05 for 1% increase, 95% confidence interval [CI]: 1.04-1.07, P < .001), serum PSA level, clinical stage, primary biopsy Gleason grade (OR = 2.52 for a grade increase, 95% CI: 1.27-5.22, P = .096), and secondary biopsy Gleason grade were independent predictors of LNI. A nomogram built using these predictive variables showed good calibration and a net clinical benefit, with an area under the curve (AUC) value of 90.2%. CONCLUSIONS: In clinical practice, the application of our nomogram might contribute significantly to the selection of patients who are good candidates for surgery with extended pelvic lymph node dissection.


Assuntos
Biomarcadores Tumorais/genética , Metástase Linfática/genética , Nomogramas , Neoplasias da Próstata/genética , Idoso , Área Sob a Curva , Proteínas de Ciclo Celular/metabolismo , Bases de Dados Genéticas , Endodesoxirribonucleases/metabolismo , Humanos , Modelos Logísticos , Linfonodos/patologia , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Análise Multivariada , Gradação de Tumores , Proteínas de Neoplasias/metabolismo , Razão de Chances , Peptídeos/metabolismo , Valor Preditivo dos Testes , Neoplasias da Próstata/patologia , Fatores de Processamento de RNA/metabolismo , Receptores Imunológicos/metabolismo , Reprodutibilidade dos Testes , Fatores de Risco , alfa Catenina/metabolismo
2.
Yi Chuan ; 41(6): 509-523, 2019 Jun 20.
Artigo em Chinês | MEDLINE | ID: mdl-31257199

RESUMO

UDP-glucuronosyltransferases (UGTs) are an important family of phase 2 drug-metabolizing enzymes that catalyze the glucuronidation of numerous endogenous or exogenous small compounds. The aberrant expression of UGT isoforms causes many diseases, such as hyperbilirubinemia and affect drug efficacy or toxicity. Understanding mechanisms of UGT gene regulation will provide scientific foundations for disease prevention and personalized or precision medicine. Vertebrate UGT family genes can be divided into UGT1 and UGT2 subfamilies. Similar to the protocadherin, immunoglobulin, and T-cell receptor gene clusters and different from the UGT2 gene cluster, the UGT1 gene cluster is organized into variable and constant regions. The UGT1 variable region contains a tandem array of variable exons, each of which can be alternatively spliced to a single set of 4 downstream constant exons, generating at least nine UGT1 mRNAs that could be translated into different UGT1 glucuronyltransferase isoforms. Our previous work reveals that the relative orientations and locations of CTCF binding sites play a key role in the three-dimensional organization of the mammalian genomes in cell nuclei. Thus in order to study the transcriptional mechanisms of UGT1 gene cluster, the distributions and orientations of CTCF binding sites (CBSs) are analyzed and compared between human and mouse UGT1 gene clusters. We find that the CBSs in the UGT1 gene cluster are not conserved between human and mouse species. We show that CTCF and cohesin regulate the transcription of the UGT1 gene cluster by knocking down the CTCF or the cohesin subunit SMC3 in the human A549 cell line. By using CRISPR DNA-fragment editing, we deleted and inverted hCBS1. By RNA-seq experiments, we find that hCBS1 deletion results in a significant decrease of levels of the UGT1A6, UGT1A7, and UGT1A9 gene expression and that hCBS1 inversion results in a significant decrease of levels of the UGT1A7 gene expression. Our data suggest that the CTCF binding site hCBS1 plays an important regulatory role in the regulation of UGT1 gene expression, providing an experimental basis for further mechanistic studies of the 3D genome regulation of the UGT1 gene cluster.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Regulação da Expressão Gênica , Glucuronosiltransferase/genética , Família Multigênica , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Éxons , Humanos , Camundongos , RNA Mensageiro
3.
Nat Commun ; 10(1): 2502, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31175280

RESUMO

Accumulation of nucleotide building blocks prior to and during S phase facilitates DNA duplication. Herein, we find that the anaphase-promoting complex/cyclosome (APC/C) synchronizes ribose-5-phosphate levels and DNA synthesis during the cell cycle. In late G1 and S phases, transketolase-like 1 (TKTL1) is overexpressed and forms stable TKTL1-transketolase heterodimers that accumulate ribose-5-phosphate. This accumulation occurs by asymmetric production of ribose-5-phosphate from the non-oxidative pentose phosphate pathway and prevention of ribose-5-phosphate removal by depleting transketolase homodimers. In the G2 and M phases after DNA synthesis, expression of the APC/C adaptor CDH1 allows APC/CCDH1 to degrade D-box-containing TKTL1, abrogating ribose-5-phosphate accumulation by TKTL1. TKTL1-overexpressing cancer cells exhibit elevated ribose-5-phosphate levels. The low CDH1 or high TKTL1-induced accumulation of ribose-5-phosphate facilitates nucleotide and DNA synthesis as well as cell cycle progression in a ribose-5-phosphate-saturable manner. Here we reveal that the cell cycle control machinery regulates DNA synthesis by mediating ribose-5-phosphate sufficiency.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdh1/metabolismo , Ciclo Celular , Replicação do DNA , Ribosemonofosfatos/metabolismo , Transcetolase/metabolismo , Proteínas Cdc20/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Fase G2 , Humanos , Via de Pentose Fosfato , Fase S
4.
Nat Commun ; 10(1): 2862, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253793

RESUMO

DNA double strand breaks (DSBs) pose a high risk for genome integrity. Cells repair DSBs through homologous recombination (HR) when a sister chromatid is available. HR is upregulated by the cycling dependent kinase (CDK) despite the paradox of telophase, where CDK is high but a sister chromatid is not nearby. Here we study in the budding yeast the response to DSBs in telophase, and find they activate the DNA damage checkpoint (DDC), leading to a telophase-to-G1 delay. Outstandingly, we observe a partial reversion of sister chromatid segregation, which includes approximation of segregated material, de novo formation of anaphase bridges, and coalescence between sister loci. We finally show that DSBs promote a massive change in the dynamics of telophase microtubules (MTs), together with dephosphorylation and relocalization of kinesin-5 Cin8. We propose that chromosome segregation is not irreversible and that DSB repair using the sister chromatid is possible in telophase.


Assuntos
Cromátides/metabolismo , Segregação de Cromossomos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Fúngico/genética , Troca de Cromátide Irmã , Telófase/genética , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Recombinação Genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Nat Commun ; 10(1): 2861, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253795

RESUMO

Centromeres provide a pivotal function for faithful chromosome segregation. They serve as a foundation for the assembly of the kinetochore complex and spindle connection, which is essential for chromosome biorientation. Cells lacking Polo-like kinase 1 (PLK1) activity suffer severe chromosome alignment defects, which is believed primarily due to unstable kinetochore-microtubule attachment. Here, we reveal a previously undescribed mechanism named 'centromere disintegration' that drives chromosome misalignment in PLK1-inactivated cells. We find that PLK1 inhibition does not necessarily compromise metaphase establishment, but instead its maintenance. We demonstrate that this is caused by unlawful unwinding of DNA by BLM helicase at a specific centromere domain underneath kinetochores. Under bipolar spindle pulling, the distorted centromeres are promptly decompacted into DNA threadlike molecules, leading to centromere rupture and whole-chromosome arm splitting. Consequently, chromosome alignment collapses. Our study unveils an unexpected role of PLK1 as a chromosome guardian to maintain centromere integrity for chromosome biorientation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fuso Acromático/fisiologia , Linhagem Celular , Pareamento Cromossômico/fisiologia , Humanos , Cinetocoros , Interferência de RNA , Timidina/farmacologia
6.
Nat Commun ; 10(1): 2612, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197141

RESUMO

Primary microcephaly is caused by mutations in genes encoding centrosomal proteins including WDR62 and KIF2A. However, mechanisms underlying human microcephaly remain elusive. By creating mutant mice and human cerebral organoids, here we found that WDR62 deletion resulted in a reduction in the size of mouse brains and organoids due to the disruption of neural progenitor cells (NPCs), including outer radial glia (oRG). WDR62 ablation led to retarded cilium disassembly, long cilium, and delayed cell cycle progression leading to decreased proliferation and premature differentiation of NPCs. Mechanistically, WDR62 interacts with and promotes CEP170's localization to the basal body of primary cilium, where CEP170 recruits microtubule-depolymerizing factor KIF2A to disassemble cilium. WDR62 depletion reduced KIF2A's basal body localization, and enhanced KIF2A expression partially rescued deficits in cilium length and NPC proliferation. Thus, modeling microcephaly with cerebral organoids and mice reveals a WDR62-CEP170-KIF2A pathway promoting cilium disassembly, disruption of which contributes to microcephaly.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinesina/metabolismo , Microcefalia/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Técnicas de Cultura de Células , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Cílios/metabolismo , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Camundongos Knockout , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/patologia , Neuroglia/citologia , Neuroglia/patologia , Organoides/patologia , Fosfoproteínas/genética , RNA Interferente Pequeno/metabolismo
7.
Nat Commun ; 10(1): 2400, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160565

RESUMO

BET-bromodomain inhibition (BETi) has shown pre-clinical promise for MYC-amplified medulloblastoma. However, the mechanisms for its action, and ultimately for resistance, have not been fully defined. Here, using a combination of expression profiling, genome-scale CRISPR/Cas9-mediated loss of function and ORF/cDNA driven rescue screens, and cell-based models of spontaneous resistance, we identify bHLH/homeobox transcription factors and cell-cycle regulators as key genes mediating BETi's response and resistance. Cells that acquire drug tolerance exhibit a more neuronally differentiated cell-state and expression of lineage-specific bHLH/homeobox transcription factors. However, they do not terminally differentiate, maintain expression of CCND2, and continue to cycle through S-phase. Moreover, CDK4/CDK6 inhibition delays acquisition of resistance. Therefore, our data provide insights about the mechanisms underlying BETi effects and the appearance of resistance and support the therapeutic use of combined cell-cycle inhibitors with BETi in MYC-amplified medulloblastoma.


Assuntos
Azepinas/farmacologia , Ciclo Celular/efeitos dos fármacos , Neoplasias Cerebelares/tratamento farmacológico , Meduloblastoma/tratamento farmacológico , Neurogênese/efeitos dos fármacos , Proteínas/antagonistas & inibidores , Triazóis/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula , Neoplasias Cerebelares/genética , Ciclina D2/efeitos dos fármacos , Ciclina D2/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos , Perfilação da Expressão Gênica , Humanos , Meduloblastoma/genética , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Fase S/efeitos dos fármacos
8.
Nat Commun ; 10(1): 2055, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-31053714

RESUMO

Autophagy is an essential recycling and quality control pathway. Mammalian ATG8 proteins drive autophagosome formation and selective removal of protein aggregates and organelles by recruiting autophagy receptors and adaptors that contain a LC3-interacting region (LIR) motif. LIR motifs can be highly selective for ATG8 subfamily proteins (LC3s/GABARAPs), however the molecular determinants regulating these selective interactions remain elusive. Here we show that residues within the core LIR motif and adjacent C-terminal region as well as ATG8 subfamily-specific residues in the LIR docking site are critical for binding of receptors and adaptors to GABARAPs. Moreover, rendering GABARAP more LC3B-like impairs autophagy receptor degradation. Modulating LIR binding specificity of the centriolar satellite protein PCM1, implicated in autophagy and centrosomal function, alters its dynamics in cells. Our data provides new mechanistic insight into how selective binding of LIR motifs to GABARAPs is achieved, and elucidate the overlapping and distinct functions of ATG8 subfamily proteins.


Assuntos
Motivos de Aminoácidos/fisiologia , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia , Ligação Proteica/fisiologia , Autoantígenos/isolamento & purificação , Autoantígenos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/isolamento & purificação , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/isolamento & purificação , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
9.
Eur J Med Chem ; 174: 265-276, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31051401

RESUMO

On the basis of the hybridization strategy of natural products, a total of 32 novel celastrol hybrids were designed, synthesized and evaluated for their antitumor activities. Most of these derivatives exihibited significant antiproliferative activities compared to celastrol, among which compound 29 displayed the strongest inhibitory capability [IC50 = 0.15 ±â€¯0.03 µM (A549),0.17 ±â€¯0.03 µM (MCF-7), 0.26 ±â€¯0.02 µM (HepG2)], which exhibited equal or superior anti-cancer activities in comparison to 2-cyano-3,12-dioxoolean-1,9 (11)-dien-28-oic acid methyl ester (CDDO-Me). The mechanism of pharmacological research indicated that 29 possessed the ability to disrupt Hsp90-Cdc37 complex which was stronger than celastrol. Meanwhile, compound 29 could induce abnormal regulation of clients (p-Akt and Cdk4) of Hsp90 and cell cycle arrest at G0/G1 phase in a concentration-dependent manner. In addition, compound 29 could also induce cell apoptosis through the death receptor pathway on A549 cells. Taken together, our results demonstrated that 29 might be a promising novel candidate for further druggability research.


Assuntos
Antineoplásicos/farmacologia , Triterpenos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Chaperoninas/metabolismo , Desenho de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Relação Estrutura-Atividade , Triterpenos/síntese química , Triterpenos/química
10.
Nan Fang Yi Ke Da Xue Xue Bao ; 39(5): 591-597, 2019 May 30.
Artigo em Chinês | MEDLINE | ID: mdl-31140425

RESUMO

OBJECTIVE: To investigate the expression of the cell division- associated gene NUF2 in breast cancer and its clinical significance. METHODS: The expression of NUF2 in breast cancer tissues was analyzed using Oncomine database. The relationship between the expression of NUF2 and the prognosis of breast cancer was analyzed using the Kaplan-Meier Plotter database. Gene set enrichment analysis (GSEA) and GEO database were used to investigate the effect of NUF2 on gene enrichment. The String database was utilized to analyze the proteins associated with NUF2. The TIMER database was analyzed to assess the correlations of NUF2 with BUB1, MAD2L1 and MYC. The expressions of NUF2 mRNA in 8 pairs of breast cancer tissues and adjacent tissues were verified by q-PCR. RESULTS: Compared with that in normal breast tissue, NUF2 was significantly overexpressed in breast cancer (P < 0.001). The overall survival time (HR = 1.52, P = 0.015) and the recurrence-free survival time (HR = 1.85, P = 3.2e-14) of the patients with high NUF2 expression were significantly shorter than those of patients with low NUF2 expression. In patients with high NUF2 expression, the enriched genes were involved mainly in cell cycle, P53, G2/M, DNA repair, MYC, and PI3K-AKT-MTOR signaling pathways, which were associated with tumor proliferation, invasion, metastasis and stemness. Combination of the results of String database, gene enrichment and TIMER database analyses suggested that NUF2 interacted directly with BUB1, MAD2L1, and MYC, which could promote the progression of breast cancer. The results of q-PCR showed that NUF2 expression was up-regulated in 6 cancer tissues and down-regulated in 2 cancer tissues. CONCLUSIONS: NUF2 gene is overexpressed in breast cancer, and its expression level is important in predicting the prognosis of breast cancer.


Assuntos
Neoplasias da Mama , Proteínas de Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Humanos , Fosfatidilinositol 3-Quinases , Prognóstico
11.
Carbohydr Polym ; 216: 97-106, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31047086

RESUMO

Here, we identified inulin-type oligosaccharides with 3-13 degrees of polymerization from Morinda officinalis. Radical-scavenging assays showed that Inulins 4-7 had modest anti-oxidative effects. Inulins 4 and 5 dose-dependently increased human umbilical vein endothelial cell survival during hypoxia/re-oxygenation (H/R)-induced injury, and Inulin 5 promoted angiogenesis. Triplicate assays with the Affymetrix Human Transcriptome Array 2.0 showed that Inulin 5 exposure up-regulated genes associated with cell cycle progression, apoptosis, DNA replication and repair, ubiquitin-mediated proteolysis, the mitogen-activated protein kinase pathway, and the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-signaling pathway. Flow cytometry, reverse transcription-quantitative polymerase chain reaction, and western blot experiments verified the microarray results and demonstrated that Inulin 5 influenced cell cycle progression and the PI3K-protein kinase B (PKB)-endothelial nitric oxide synthase (eNOS) pathway. Thus, inulin-type oligosaccharides from M. officinalis roots may protect against H/R-induced injury, primarily through an anti-oxidative effect, and promote angiogenesis by activating the PI3K-PKB-eNOS-signaling pathway.


Assuntos
Depuradores de Radicais Livres/farmacologia , Hipóxia/tratamento farmacológico , Inulina/farmacologia , Morinda/química , Oligossacarídeos/farmacologia , Indutores da Angiogênese/isolamento & purificação , Indutores da Angiogênese/farmacologia , Sequência de Carboidratos , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Depuradores de Radicais Livres/isolamento & purificação , Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Inulina/isolamento & purificação , Peso Molecular , Óxido Nítrico Sintase Tipo III/metabolismo , Oligossacarídeos/isolamento & purificação , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos
12.
Int J Mol Sci ; 20(9)2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-31035388

RESUMO

Transactivation of p21 (cyclin-dependent kinase inhibitor 1A, CDKN1A) is closely related to the recruitment of transcription cofactors at the p53 responsive elements (p53REs) in its promoter region. Human chromatin remodeling enzyme INO80 can be recruited to the p53REs of p21 promoter and negatively regulates p21. As one of the key subunits of the INO80 complex, YY1 has also been confirmed to bind to the p53RE sites of p21 promoter. Importantly, YY1 was recently reported to be bound and stabilized by BCCIP (BRCA2 and CDKN1A-interacting protein). Therefore, we hypothesized that the YY1/BCCIP complex plays an important role in regulating the transactivation of p21. Here we present evidence that the YY1/BCCIP complex coordinatively regulates p53RE-mediated p21 transactivation. We first confirmed the cross-interaction between YY1, BCCIP, and p53, suggesting an intrinsic link between three proteins in the regulation of p21 transcription. In dual luciferase assays, YY1 inhibited p53RE-mediated luciferase activity, whereas BCCIP revealed the opposite effect. More interestingly, the region 146-270 amino acids of YY1, which bound to BCCIP, increased p53-mediated luciferase activity, indicating the complexity of the YY1/BCCIP complex in co-regulating p21 transcription. Further in-depth research confirmed the co-occupancy of YY1/BCCIP with p53 at the p53RE-proximal region of p21. Lentiviral-mediated knockdown of BCCIP inhibited the recruitment of p53 and YY1 at the p53RE proximal region of p21; however, this phenomenon was reversed by expressing exogenous YY1, suggesting the collaborative regulation of YY1/BCCIP complex in p53RE-mediated p21 transcription. These data provide new insights into the transcriptional regulation of p21 by the YY1/BCCIP complex.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteínas Nucleares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fator de Transcrição YY1/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Células HCT116 , Humanos , Proteínas Nucleares/genética , Ativação Transcricional/genética , Ativação Transcricional/fisiologia , Fator de Transcrição YY1/genética
13.
Nat Commun ; 10(1): 1525, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30948724

RESUMO

Essentially all cellular processes are orchestrated by protein-protein interactions (PPIs). In recent years, affinity purification coupled to mass spectrometry (AP-MS) has been the preferred method to identify cellular PPIs. Here we present a microfluidic-based AP-MS workflow, called on-chip AP-MS, to identify PPIs using minute amounts of input material. By using this automated platform we purify the human Cohesin, CCC and Mediator complexes from as little as 4 micrograms of input lysate, representing a 50─100-fold downscaling compared to regular microcentrifuge tube-based protocols. We show that our platform can be used to affinity purify tagged baits as well as native cellular proteins and their interaction partners. As such, our method holds great promise for future biological and clinical AP-MS applications in which sample amounts are limited.


Assuntos
Microfluídica/métodos , Mapas de Interação de Proteínas , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Humanos , Complexo Mediador/metabolismo , Microfluídica/instrumentação , Purificação por Afinidade em Tandem , Espectrometria de Massas em Tandem
14.
Nat Commun ; 10(1): 1526, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30948730

RESUMO

In eukaryotes, adventitious oxidation of erythrose-4-phosphate, an intermediate of the pentose phosphate pathway (PPP), generates 4-phosphoerythronate (4PE), which inhibits 6-phosphogluconate dehydrogenase. 4PE is detoxified by metabolite-proofreading phosphatases such as yeast Pho13. Here, we report that a similar function is carried out in Bacillus subtilis by CpgA, a checkpoint protein known to be important for ribosome assembly, cell morphology and resistance to cell wall-targeting antibiotics. We find that ΔcpgA cells are intoxicated by glucose or other carbon sources that feed into the PPP, and that CpgA has high phosphatase activity with 4PE. Inhibition of 6-phosphogluconate dehydrogenase (GndA) leads to intoxication by 6-phosphogluconate, a potent inhibitor of phosphoglucose isomerase (PGI). The coordinated shutdown of PPP and glycolysis leads to metabolic gridlock. Overexpression of GndA, PGI, or yeast Pho13 suppresses glucose intoxication of ΔcpgA cells, but not cold sensitivity, a phenotype associated with ribosome assembly defects. Our results suggest that CpgA is a multifunctional protein, with genetically separable roles in ribosome assembly and metabolite proofreading.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/fisiologia , Proteínas de Ciclo Celular/fisiologia , Monoéster Fosfórico Hidrolases/fisiologia , Ribossomos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Glicólise , Modelos Moleculares , Via de Pentose Fosfato , Fosfogluconato Desidrogenase/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Açúcares Ácidos/metabolismo
15.
Cell Mol Biol (Noisy-le-grand) ; 65(3): 41-47, 2019 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-30942154

RESUMO

Recently the role of indole and pyran rings in carcinogenesis has been well studied. Here we studied the effects and the possible mechanisms of the action of basal indole (I3A) and its novel indole derivative (C19H15F3N2O) on inhibition of proliferation cells in acute promyelocytic leukemia NB4 cell line by examining the expression of cell cycle genes. We treated NB4 cells with concentration of C19H15F3N2O for 24-72 h. The MTT and PI/Annexin V examinations were employed for assessment of the proliferation and apoptosis of NB4 cells. Both of Cyclin D and P21 were detected by the Real-time PCR. The western blotting analysis was also performed to show the protein levels for P21. A difference was regarded significant if p-value was less than 0.05. MTT assay showed that 15.12-1000 µg/mL C19H15F3N2O caused a time and concentration-dependent inhibition of NB4 cell proliferation. Exposure to higher concentrations of C19H15F3N2O resulted in significantly increased apoptosis rate in NB4 cells. RT PCR showed that C19H15F3N2O has up-regulated the expression of P21 and down-regulated the expression of Cyclin D. Western blotting experiments also demonstrated that the P21 expression in C19H15F3N2O treated cells has significantly increased, where compared with either untreated control cells or I3A treated cells. This newly (C19H15F3N2O) was able to inhibit NB4 cells proliferation and causes apoptosis of these cells more than I3A, and these effects are probably facilitated via cell cycle arrest. C19H15F3N2O might probably be introduced as a promising organic therapeutic reagent against APL.


Assuntos
Proteínas de Ciclo Celular/genética , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Indóis/farmacologia , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Indóis/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
16.
Nat Commun ; 10(1): 1810, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-31000710

RESUMO

During centriole duplication, a single daughter centriole is formed next to the mother centriole. The molecular mechanism that determines a single duplication site remains a long-standing question. Here, we show that intrinsic self-organization of Plk4 is implicated in symmetry breaking in the process of centriole duplication. We demonstrate that Plk4 has an ability to phase-separate into condensates via an intrinsically disordered linker and that the condensation properties of Plk4 are regulated by autophosphorylation. Consistently, the dissociation dynamics of centriolar Plk4 are controlled by autophosphorylation. We further found that autophosphorylated Plk4 is already distributed as a single focus around the mother centriole before the initiation of procentriole formation, and is subsequently targeted for STIL-HsSAS6 loading. Perturbation of Plk4 self-organization affects the asymmetry of centriolar Plk4 distribution and proper centriole duplication. Overall, we propose that the spatial pattern formation of Plk4 is a determinant of a single duplication site per mother centriole.


Assuntos
Ciclo Celular/fisiologia , Centríolos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microscopia Intravital/métodos , Microscopia Confocal/métodos , Fosforilação/efeitos dos fármacos , Agregados Proteicos/fisiologia , Domínios Proteicos/fisiologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/isolamento & purificação , Pirimidinas/farmacologia , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sulfonas/farmacologia
17.
Cancer Sci ; 110(6): 1897-1908, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31006167

RESUMO

Xp11.2 translocation renal cell carcinoma (Xp11 tRCC) is a rare sporadic pediatric kidney cancer caused by constitutively active TFE3 fusion proteins. Tumors in patients with Xp11 tRCC tend to recur and undergo frequent metastasis, in part due to lack of methods available to detect early-stage disease. Here we generated transgenic (Tg) mice overexpressing the human PRCC-TFE3 fusion gene in renal tubular epithelial cells, as an Xp11 tRCC mouse model. At 20 weeks of age, mice showed no histological abnormalities in kidney but by 40 weeks showed Xp11 tRCC development and related morphological and histological changes. MicroRNA (miR)-204-5p levels in urinary exosomes of 40-week-old Tg mice showing tRCC were significantly elevated compared with levels in control mice. MicroRNA-204-5p expression also significantly increased in primary renal cell carcinoma cell lines established both from Tg mouse tumors and from tumor tissue from 2 Xp11 tRCC patients. All of these lines secreted miR-204-5p-containing exosomes. Notably, we also observed increased miR-204-5p levels in urinary exosomes in 20-week-old renal PRCC-TFE3 Tg mice prior to tRCC development, and those levels were equivalent to those in 40-week-old Tg mice, suggesting that miR-204-5p increases follow expression of constitutively active TFE3 fusion proteins in renal tubular epithelial cells prior to overt tRCC development. Finally, we confirmed that miR-204-5p expression significantly increases in noncancerous human kidney cells after overexpression of a PRCC-TFE3 fusion gene. These findings suggest that miR-204-5p in urinary exosomes could be a useful biomarker for early diagnosis of patients with Xp11 tRCC.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Cromossomos Humanos X/genética , Neoplasias Renais/genética , MicroRNAs/genética , Translocação Genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Biomarcadores Tumorais/urina , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/urina , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Exossomos/genética , Humanos , Rim/anormalidades , Rim/metabolismo , Neoplasias Renais/metabolismo , Neoplasias Renais/urina , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/urina , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo
18.
Nat Commun ; 10(1): 1577, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30952868

RESUMO

DNA double-strand break (DSB) signaling and repair are critical for genome integrity. They rely on highly coordinated processes including posttranslational modifications of proteins. Here we show that Pellino1 (Peli1) is a DSB-responsive ubiquitin ligase required for the accumulation of DNA damage response proteins and efficient homologous recombination (HR) repair. Peli1 is activated by ATM-mediated phosphorylation. It is recruited to DSB sites in ATM- and γH2AX-dependent manners. Interaction of Peli1 with phosphorylated histone H2AX enables it to bind to and mediate the formation of K63-linked ubiquitination of NBS1, which subsequently results in feedback activation of ATM and promotes HR repair. Collectively, these results provide a DSB-responsive factor underlying the connection between ATM kinase and DSB-induced ubiquitination.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Humanos , Proteínas Nucleares/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
19.
Epigenetics Chromatin ; 12(1): 24, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992049

RESUMO

BACKGROUND: Histone acetylation plays an important role in DNA replication and repair because replicating chromatin is subject to dynamic changes in its structures. However, its precise mechanism remains elusive. In this report, we describe roles of the NuA4 acetyltransferase and histone H4 acetylation in replication fork protection in the fission yeast Schizosaccharomyces pombe. RESULTS: Downregulation of NuA4 subunits renders cells highly sensitive to camptothecin, a compound that induces replication fork breakage. Defects in NuA4 function or mutations in histone H4 acetylation sites lead to impaired recovery of collapsed replication forks and elevated levels of Rad52 DNA repair foci, indicating the role of histone H4 acetylation in DNA replication and fork repair. We also show that Vid21 interacts with the Swi1-Swi3 replication fork protection complex and that Swi1 stabilizes Vid21 and promotes efficient histone H4 acetylation. Furthermore, our genetic analysis demonstrates that loss of Swi1 further sensitizes NuA4 and histone H4 mutant cells to replication fork breakage. CONCLUSION: Considering that Swi1 plays a critical role in replication fork protection, our results indicate that NuA4 and histone H4 acetylation promote repair of broken DNA replication forks.


Assuntos
Replicação do DNA , Histona Acetiltransferases/metabolismo , Acetilação , Camptotecina/toxicidade , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histona Acetiltransferases/genética , Histonas/genética , Histonas/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Inibidores da Topoisomerase I/toxicidade
20.
Chem Biol Interact ; 305: 112-118, 2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-30935902

RESUMO

Previous studies have demonstrated that different curcumin extracts are able to influence cell metabolic activity vitality in human papillary thyroid carcinoma TPC-1 cells. We continued the study using the most effective extract and adding other nutraceuticals such as piperine and vitamin E, in order to define the possible role of these in modulating the genetic expression of cell markers and to understand the effectiveness in modulating the regression of cancer phenotype. Cells were treated with one extract of curcumin (Naturex® Ultimate Botanical Benefits), with Piperine (Piper Longum, A.C.E.F.) and Vitamin E (Dry Vitamin E-Acetate 50% DC, BASF) alone and in combination, dissolved in the culture medium, for 48 h. Treatment with the different nutraceuticals is able to influence cell cycle regulators (cyclin D1, ß-catenin, p21, p53) and activators or inhibitors of apoptosis (BAX, pro-caspase3, Bcl-2). They are able to influence cell cycle distribution and metabolic activity vitality. The inhibitory effect of curcumin, piperine and vitamin E on cell proliferation involves different markers, and in particular inhibits ß-catenin, cyclinD1 and p53, making them candidates for a possible use in alternative therapies although further studies are needed.


Assuntos
Proliferação de Células/efeitos dos fármacos , Curcumina/farmacologia , Alcaloides/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Benzodioxóis/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Câncer Papilífero da Tireoide/metabolismo , Câncer Papilífero da Tireoide/patologia , Vitamina E/farmacologia
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