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1.
PLoS One ; 15(10): e0235103, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33075068

RESUMO

PCNA sliding clamp binds factors through which histone deposition, chromatin remodeling, and DNA repair are coupled to DNA replication. PCNA also directly binds Eco1/Ctf7 acetyltransferase, which in turn activates cohesins and establishes cohesion between nascent sister chromatids. While increased recruitment thus explains the mechanism through which elevated levels of chromatin-bound PCNA rescue eco1 mutant cell growth, the mechanism through which PCNA instead worsens cohesin mutant cell growth remains unknown. Possibilities include that elevated levels of long-lived chromatin-bound PCNA reduce either cohesin deposition onto DNA or cohesin acetylation. Instead, our results reveal that PCNA increases the levels of both chromatin-bound cohesin and cohesin acetylation. Beyond sister chromatid cohesion, PCNA also plays a critical role in genomic stability such that high levels of chromatin-bound PCNA elevate genotoxic sensitivities and recombination rates. At a relatively modest increase of chromatin-bound PCNA, however, fork stability and progression appear normal in wildtype cells. Our results reveal that even a moderate increase of PCNA indeed sensitizes cohesin mutant cells to DNA damaging agents and in a process that involves the DNA damage response kinase Mec1(ATR), but not Tel1(ATM). These and other findings suggest that PCNA mis-regulation results in genome instabilities that normally are resolved by cohesin. Elevating levels of chromatin-bound PCNA may thus help target cohesinopathic cells linked that are linked to cancer.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , DNA Fúngico/genética , Instabilidade Genômica , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Acetilação , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
2.
Cell Prolif ; 53(10): e12908, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32951278

RESUMO

OBJECTIVES: In this study, we comprehensively analysed the role of ubiquitin-specific protease 1(USP1) in hepatocellular carcinoma (HCC) using data from a set of public databases. MATERIALS AND METHODS: We analysed the mRNA expression of USP1 in HCC and subgroups of HCC using Oncomine and UALCAN. Survival analysis of USP1 in HCC was conducted with the Kaplan-Meier Plotter database. The mutations of USP1 in HCC were analysed using cBioPortal and the Catalogue of Somatic Mutations in Cancer database. Differential genes correlated with USP1 and WD repeat domain 48 (WDR48) were obtained using LinkedOmics. USP1 was knocked down with small interfering RNA (siRNA) or pharmacologically inhibited by ML-323 in MHCC97H or SK-Hep-1 cell lines for function analysis. RESULTS: High USP1 expression predicted unfavourable overall survival in HCC patients. USP1 showed positive correlations with the abundances of macrophages and neutrophils. We identified 98 differential genes that were positively correlated with both USP1 and WDR48. These genes were mainly involved in the cell cycle, aldosterone synthesis and secretion and oestrogen signalling pathways. Interactions between USP1 and WDR 48 were confirmed using co-immunoprecipitation. USP1 knockdown or ML-323 treatment reduced the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E1. CONCLUSIONS: Overall, USP1 is a promising target for HCC treatment with good prognostic value. USP1 and WDR48 function together in regulating cancer cell proliferation via the cell cycle.


Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteases Específicas de Ubiquitina/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Intervalo Livre de Doença , Inibidores Enzimáticos/farmacologia , Estrogênios/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Prognóstico , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Mapas de Interação de Proteínas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/genética
3.
Proc Natl Acad Sci U S A ; 117(38): 23588-23596, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32900933

RESUMO

In human cells, the DNA replication factor proliferating cell nuclear antigen (PCNA) can be conjugated to either the small ubiquitinlike modifier SUMO1 or SUMO2, but only SUMO2-conjugated PCNA is induced by transcription to facilitate resolution of transcription-replication conflict (TRC). To date, the SUMO E3 ligase that provides substrate specificity for SUMO2-PCNA conjugation in response to TRC remains unknown. Using a proteomic approach, we identified TRIM28 as the E3 ligase that catalyzes SUMO2-PCNA conjugation. In vitro, TRIM28, together with the RNA polymerase II (RNAPII)-interacting protein RECQ5, promotes SUMO2-PCNA conjugation but inhibits SUMO1-PCNA formation. This activity requires a PCNA-interacting protein (PIP) motif located within the bromodomain of TRIM28. In cells, TRIM28 interaction with PCNA on human chromatin is dependent on both transcription and RECQ5, and SUMO2-PCNA level correlates with TRIM28 expression. As a consequence, TRIM28 depletion led to RNAPII accumulation at TRC sites, and expression of a TRIM28 PIP mutant failed to suppress TRC-induced DNA breaks.


Assuntos
Replicação do DNA/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteína 28 com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Quebras de DNA , Células HEK293 , Humanos , Antígeno Nuclear de Célula em Proliferação/genética , RecQ Helicases/genética , RecQ Helicases/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteína 28 com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética
4.
J Card Surg ; 35(8): 1912-1919, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32652694

RESUMO

BACKGROUNDS: Disparities may exist between the adolescent and the adult patients with cardiac fibromas in the symptoms, surgical outcomes, and pathological characteristics. The aim of this study was to compare short and midterm surgical outcomes of cardiac fibromas and to compare the biomarker expressions of tumor tissue samples between the adult and the adolescent. METHODS: Consecutive patients with the diagnosis of cardiac fibroma were admitted and received surgeries. Primary outcomes included in-hospital mortality, low cardiac output, and readmission due to heart failure. The expression of PCNA and Ki67, two widely adopted indicators of cell proliferation, were evaluated in tissue samples. RESULTS: A total of five adolescent patients and five adult patients diagnosed as cardiac fibroma were admitted and given surgeries. When compare with the adults, the adolescent patients were more likely to present symptoms on admission (P = .048). Postoperative low cardiac output syndrome was significantly higher in the adolescents than in the adults (80.0% vs 0.0, P = .048). The tumor volume relative to ventricular end diastolic diameter had good discriminative ability for low cardiac output (c statistics: 0.96). Pathologically, the percentage of PCNA-positive cell nuclei was significantly higher in the adolescents than in the adults (36.04% ± 10.54% vs 4.15% ± 3.93%, P = .001). However, there were no Ki67-positive nuclei in the 10 cases. CONCLUSIONS: In the current study, we found that postoperative low cardiac output was more likely to occur in the adolescent patients than in the adult patients. When compared with the adult patients, significantly more PCNA-positive nuclei were observed in the adolescents.


Assuntos
Procedimentos Cirúrgicos Cardíacos , Fibroma/patologia , Fibroma/cirurgia , Neoplasias Cardíacas/patologia , Neoplasias Cardíacas/cirurgia , Adolescente , Adulto , Fatores Etários , Baixo Débito Cardíaco/epidemiologia , Proliferação de Células/genética , Feminino , Expressão Gênica , Humanos , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/epidemiologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Resultado do Tratamento , Adulto Jovem
5.
Nat Commun ; 11(1): 3664, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694532

RESUMO

Ethanol is a ubiquitous environmental stressor that is toxic to all lifeforms. Here, we use the model eukaryote Saccharomyces cerevisiae to show that exposure to sublethal ethanol concentrations causes DNA replication stress and an increased mutation rate. Specifically, we find that ethanol slows down replication and affects localization of Mrc1, a conserved protein that helps stabilize the replisome. In addition, ethanol exposure also results in the recruitment of error-prone DNA polymerases to the replication fork. Interestingly, preventing this recruitment through mutagenesis of the PCNA/Pol30 polymerase clamp or deleting specific error-prone polymerases abolishes the mutagenic effect of ethanol. Taken together, this suggests that the mutagenic effect depends on a complex mechanism, where dysfunctional replication forks lead to recruitment of error-prone polymerases. Apart from providing a general mechanistic framework for the mutagenic effect of ethanol, our findings may also provide a route to better understand and prevent ethanol-associated carcinogenesis in higher eukaryotes.


Assuntos
Replicação do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Etanol/toxicidade , Taxa de Mutação , Saccharomyces cerevisiae/genética , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/metabolismo , DNA Fúngico/genética , Mutagênese , Testes de Mutagenicidade , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
Arch Biochem Biophys ; 688: 108404, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32416101

RESUMO

Hemangioma (HA) is the most common benign tumor and formed by the proliferating endothelial cells of blood vessels. Interleukins (ILs) have been reported to be critical for HA progression. Our present study found that the expression of IL-10 was decreased in HA cells and tissues as compared to their corresponding controls. Treatment with recombinant IL-10 (rIL-10) can suppress the proliferation of HA cells via suppression of proliferating cell nuclear antigen (PCNA), while over expression of PCNA can attenuate rIL-10-inhibited cell proliferation. Further, rIL-10 can decrease the promoter activity and mRNA stability of PCNA in HA cells. Mechanistically, rIL-10 can increase expression of miR-27b-3p to decrease mRNA stability of PCNA, while down regulation of YY1 is involved in rIL-10 suppressed transcription of PCNA. Collectively, IL-10 can suppress the expression of PCNA via miR-27b-3p mediated suppression of mRNA stability and YY1 mediated down regulation of transcription. It suggested that rIL-10 might be a potential therapeutic approach for HA development and progression.


Assuntos
Células Endoteliais/metabolismo , Hemangioma/prevenção & controle , Interleucina-10/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proliferação de Células/fisiologia , Progressão da Doença , Hemangioma/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Interleucina-10/metabolismo , MicroRNAs/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , Fator de Transcrição YY1/metabolismo
7.
PLoS Pathog ; 16(5): e1008190, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32413071

RESUMO

DNA replication protein Cdc45 is an integral part of the eukaryotic replicative helicase whose other components are the Mcm2-7 core, and GINS. We identified a PIP box motif in Leishmania donovani Cdc45. This motif is typically linked to interaction with the eukaryotic clamp proliferating cell nuclear antigen (PCNA). The homotrimeric PCNA can potentially bind upto three different proteins simultaneously via a loop region present in each monomer. Multiple binding partners have been identified from among the replication machinery in other eukaryotes, and the concerted /sequential binding of these partners are central to the fidelity of the replication process. Though conserved in Cdc45 across Leishmania species and Trypanosoma cruzi, the PIP box is absent in Trypanosoma brucei Cdc45. Here we investigate the possibility of Cdc45-PCNA interaction and the role of such an interaction in the in vivo context. Having confirmed the importance of Cdc45 in Leishmania DNA replication we establish that Cdc45 and PCNA interact stably in whole cell extracts, also interacting with each other directly in vitro. The interaction is mediated via the Cdc45 PIP box. This PIP box is essential for Leishmania survival. The importance of the Cdc45 PIP box is also examined in Schizosaccharomyces pombe, and it is found to be essential for cell survival here as well. Our results implicate a role for the Leishmania Cdc45 PIP box in recruiting or stabilizing PCNA on chromatin. The Cdc45-PCNA interaction might help tether PCNA and associated replicative DNA polymerase to the DNA template, thus facilitating replication fork elongation. Though multiple replication proteins that associate with PCNA have been identified in other eukaryotes, this is the first report demonstrating a direct interaction between Cdc45 and PCNA, and while our analysis suggests the interaction may not occur in human cells, it indicates that it may not be confined to trypanosomatids.


Assuntos
Leishmania donovani/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Cromatina/genética , DNA Helicases/metabolismo , Replicação do DNA/fisiologia , Leishmania donovani/genética , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Nucleotidiltransferases/genética , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/fisiologia , Análise de Sequência de Proteína/métodos
8.
Nat Commun ; 11(1): 2147, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358495

RESUMO

Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing 293T and RPE1 cells deficient in PCNA ubiquitination, generated through CRISPR/Cas9 gene editing, here, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-dependent but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork and incomplete Okazaki fragment maturation, which interferes with efficient PCNA unloading by ATAD5 and subsequent nucleosome deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and the BRCA pathway results in increased nascent DNA degradation and PARP inhibitor sensitivity. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors.


Assuntos
Antígeno Nuclear de Célula em Proliferação/metabolismo , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/fisiologia , Ensaio Cometa , DNA/genética , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , Replicação do DNA/genética , Replicação do DNA/fisiologia , Imunofluorescência , Instabilidade Genômica/genética , Instabilidade Genômica/fisiologia , Células HEK293 , Células HeLa , Humanos , Antígeno Nuclear de Célula em Proliferação/genética , Ligação Proteica , Ubiquitinação/genética , Ubiquitinação/fisiologia
9.
Nat Struct Mol Biol ; 27(5): 461-471, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32341532

RESUMO

The coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Disruption of the balance between helicase and polymerase activities during replication stress leads to fork progression defects and activation of the Rad53 checkpoint kinase, which is essential for the functional maintenance of stalled replication forks. The mechanism of Rad53-dependent fork stabilization is not known. Using reconstituted budding yeast replisomes, we show that mutational inactivation of the leading strand DNA polymerase, Pol ε, dNTP depletion, and chemical inhibition of DNA polymerases cause excessive DNA unwinding by the replicative DNA helicase, CMG, demonstrating that budding yeast replisomes lack intrinsic mechanisms that control helicase-polymerase coupling at the fork. Importantly, we find that the Rad53 kinase restricts excessive DNA unwinding at replication forks by limiting CMG helicase activity, suggesting a mechanism for fork stabilization by the replication checkpoint.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , DNA Fúngico/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , DNA Helicases/genética , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Primers do DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/genética , Plasmídeos , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
10.
Nat Struct Mol Biol ; 27(5): 450-460, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32341533

RESUMO

Leading-strand template aberrations cause helicase-polymerase uncoupling and impede replication fork progression, but the details of how uncoupled forks are restarted remain uncertain. Using purified proteins from Saccharomyces cerevisiae, we have reconstituted translesion synthesis (TLS)-mediated restart of a eukaryotic replisome following collision with a cyclobutane pyrimidine dimer. We find that TLS functions 'on the fly' to promote resumption of rapid replication fork rates, despite lesion bypass occurring uncoupled from the Cdc45-MCM-GINS (CMG) helicase. Surprisingly, the main lagging-strand polymerase, Pol δ, binds the leading strand upon uncoupling and inhibits TLS. Pol δ is also crucial for efficient recoupling of leading-strand synthesis to CMG following lesion bypass. Proliferating cell nuclear antigen monoubiquitination positively regulates TLS to overcome Pol δ inhibition. We reveal that these mechanisms of negative and positive regulation also operate on the lagging strand. Our observations have implications for both fork restart and the division of labor during leading-strand synthesis generally.


Assuntos
Replicação do DNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Acetiltransferases/genética , Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinação
11.
Cardiovasc Ther ; 2020: 1926249, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32328171

RESUMO

Isoliquiritigenin (ISL) is a flavonoid isolated mainly from the licorice plant, a traditional Chinese herb. ISL has shown anticancer, anti-inflammatory, antioxidant, and antidiabetic activities. However, the pharmaceutical effects of ISL on atherosclerosis are seldom explored. In this study, we used apolipoprotein E (ApoE) knockout mouse model and angiotensin II- (Ang II-) stimulated vascular smooth muscle cells (VSMCs) to elucidate the pharmacological mechanism of ISL to inhibit atherosclerosis. We found that in ApoE-/- mice ISL could attenuate atherosclerotic lesion, reduce serum lipid levels, and inhibit TRPC5 expression. In vitro, ISL inhibited Ang II-stimulated proliferation of VSMCs and suppressed Ang II-induced TRPC5 and PCNA expressions in a dose-dependent fashion. In conclusion, our findings provide novel insight into the pharmacological effects of ISL on atherosclerosis and suggest that ISL is beneficial for cardiovascular protection.


Assuntos
Aorta/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Chalconas/farmacologia , Placa Aterosclerótica , Canais de Cátion TRPC/antagonistas & inibidores , Animais , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Lipídeos/sangue , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Transdução de Sinais , Canais de Cátion TRPC/metabolismo
12.
Mol Cell ; 78(4): 725-738.e4, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32277910

RESUMO

Concomitant with DNA replication, the chromosomal cohesin complex establishes cohesion between newly replicated sister chromatids. Several replication-fork-associated "cohesion establishment factors," including the multifunctional Ctf18-RFC complex, aid this process in as yet unknown ways. Here, we show that Ctf18-RFC's role in sister chromatid cohesion correlates with PCNA loading but is separable from its role in the replication checkpoint. Ctf18-RFC loads PCNA with a slight preference for the leading strand, which is dispensable for DNA replication. Conversely, the canonical Rfc1-RFC complex preferentially loads PCNA onto the lagging strand, which is crucial for DNA replication but dispensable for sister chromatid cohesion. The downstream effector of Ctf18-RFC is cohesin acetylation, which we place toward a late step during replication maturation. Our results suggest that Ctf18-RFC enriches and balances PCNA levels at the replication fork, beyond the needs of DNA replication, to promote establishment of sister chromatid cohesion and possibly other post-replicative processes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromátides/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/fisiologia , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Acetiltransferases/genética , Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
13.
Oncogene ; 39(19): 3879-3892, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32203162

RESUMO

Mutants in the gene encoding mitochondrion-associated protein LRPPRC were found to be associated with French Canadian Type Leigh syndrome, a human disorder characterized with neurodegeneration and cytochrome c oxidase deficiency. LRPPRC interacts with one of microtubule-associated protein family MAP1S that promotes autophagy initiation and maturation to suppress genomic instability and tumorigenesis. Previously, although various studies have attributed LRPPRC nuclear acid-associated functions, we characterized that LRPPRC acted as an inhibitor of autophagy in human cancer cells. Here we show that liver-specific deletion of LRPPRC causes liver-specific increases of YAP and P27 and decreases of P62, leading to an increase of cell polyploidy and an impairment of autophagy maturation. The blockade of autophagy maturation and promotion of polyploidy caused by LRPPRC depletion synergistically enhances diethylnitrosamine-induced DNA damage, genome instability, and further tumorigenesis so that LRPPRC knockout mice develop more and larger hepatocellular carcinomas and survive a shorter lifespan. Therefore, LRPPRC suppresses genome instability and hepatocellular carcinomas and promotes survivals in mice by sustaining Yap-P27-mediated cell ploidy and P62-HDAC6-controlled autophagy maturation.


Assuntos
Carcinoma Hepatocelular/genética , Deficiência de Citocromo-c Oxidase/genética , Desacetilase 6 de Histona/genética , Doença de Leigh/genética , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Autofagia/genética , Canadá , Carcinogênese/genética , Carcinoma Hepatocelular/patologia , Deficiência de Citocromo-c Oxidase/patologia , Instabilidade Genômica/genética , Células HeLa , Humanos , Doença de Leigh/patologia , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , Ploidias , Antígeno Nuclear de Célula em Proliferação/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética
14.
Life Sci ; 248: 117445, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32081664

RESUMO

AIMS: Atherosclerosis (AS) is a common cardiovascular disease with complicated pathogenesis. Long non-coding RNAs (lncRNAs) have been reported to be associated with AS progression. We aimed to explore the role and underlying mechanism of HOXA transcript at the distal tip (HOTTIP) in AS. MATERIALS AND METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of HOTTIP, miR-490-3p and high mobility group B 1 (HMGB1) in AS patients' sera and oxidized low-density lipoprotein (ox-LDL) induced human aortic vascular smooth muscle cells (HA-VSMCs). Cell Counting Kit-8 (CCK-8) assay and transwell assay were conducted to evaluate the proliferation and migration of HA-VSMCs, respectively. Western blot assay was carried out to determine the levels of proliferating cell nuclear antigen (PCNA), matrix metalloprotein 2 (MMP2), MMP9 and HMGB1. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were conducted to verify the targeting association between HOTTIP and miR-490-3p, as well as miR-490-3p and HMGB1. KEY FINDINGS: HOTTIP and HMGB1 were upregulated and miR-490-3p was downregulated in the sera of AS patients and ox-LDL-stimulated HA-VSMCs. HOTTIP knockdown suppressed ox-LDL induced proliferation and migration in HA-VSMCs. MiR-490-3p was identified as a target of HOTTIP and HOTTIP overexpression abolished the inhibition on cell proliferation and migration mediated by miR-490-3p in ox-LDL-induced HA-VSMCs. Moreover, miR-490-3p inhibition promoted cell proliferation and migration by directly targeting HMGB1 in ox-LDL-induced HA-VSMCs. Besides, HOTTIP knockdown repressed the activation of PI3K-AKT signaling pathway. SIGNIFICANCE: HOTTIP knockdown suppressed cell proliferation and migration by regulating miR-490-3p/HMGB1 axis and PI3K-AKT pathway in ox-LDL-induced HA-VSMCs.


Assuntos
Aterosclerose/genética , Proteína HMGB1/genética , MicroRNAs/genética , Miócitos de Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Longo não Codificante/genética , Aorta/metabolismo , Aorta/patologia , Aterosclerose/sangue , Aterosclerose/patologia , Estudos de Casos e Controles , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica , Proteína HMGB1/metabolismo , Humanos , Lipoproteínas LDL/farmacologia , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
15.
Nucleic Acids Res ; 48(6): 3042-3052, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32009145

RESUMO

Ubiquitylation of the eukaryotic sliding clamp, PCNA, activates a pathway of DNA damage bypass that facilitates the replication of damaged DNA. In its monoubiquitylated form, PCNA recruits a set of damage-tolerant DNA polymerases for translesion synthesis. Alternatively, modification by K63-linked polyubiquitylation triggers a recombinogenic process involving template switching. Despite the identification of proteins interacting preferentially with polyubiquitylated PCNA, the molecular function of the chain and the relevance of its K63-linkage are poorly understood. Using genetically engineered mimics of polyubiquitylated PCNA, we have now examined the properties of the ubiquitin chain required for damage bypass in budding yeast. By varying key parameters such as the geometry of the junction, cleavability and capacity for branching, we demonstrate that either the structure of the ubiquitin-ubiquitin junction or its dynamic assembly or disassembly at the site of action exert a critical impact on damage bypass, even though known effectors of polyubiquitylated PCNA are not strictly linkage-selective. Moreover, we found that a single K63-junction supports substantial template switching activity, irrespective of its attachment site on PCNA. Our findings provide insight into the interrelationship between the two branches of damage bypass and suggest the existence of a yet unidentified, highly linkage-selective receptor of polyubiquitylated PCNA.


Assuntos
Dano ao DNA/genética , Proteínas de Ligação a DNA/genética , Antígeno Nuclear de Célula em Proliferação/genética , Ubiquitinação/genética , Reparo do DNA/genética , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , Poliubiquitina/genética , Mapas de Interação de Proteínas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/genética
16.
Anim Reprod Sci ; 213: 106276, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31987327

RESUMO

There is production of prostaglandin F2α (PGF2α) and there is PGF2α receptor (PTGFR) mRNA transcript in endometrial epithelial cells of cattle. The aims of the present study were to (1) determine whether PGF2α-PTGFR signaling modulates the proliferation of endometrial epithelial cells and (2) increase knowledge of PGF2α-PTGFR signaling on the physiological and pharmacological processes in the endometrium of cattle. Amount of cellular proliferation was determined using real-time cell analysis and cell proliferation reagent WST-1 procedures. Abundance of cyclins, cyclin-dependent kinases (CDKs), cyclin-kinase inhibitors, proliferating cell nuclear antigen (PCNA), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), PTGFR, epidermal growth factor (EGF) mRNA and protein abundances were evaluated using real-time RT-PCR and western blot analyses. The PGF2α-PTGFR signaling promoted the proliferation of endometrial epithelial cells by inducing changes in abundance of mRNA transcript and protein that resulted in an increase in the abundance for the cyclins (A, B1, D1, D3), CDKs (1, 2, 4, 6), and PCNA; decrease in abundance for p21; and increase in abundance for EGF, COX-1, COX-2, and PTGFR. There was a direct molecular association between PGF2α-PTGFR signaling and cell cycle regulation in endometrial epithelial cells of cattle. In addition, findings improve the understanding of PGF2α-PTGFR signaling in the physiological and pharmacological processes of the endometrium of cattle.


Assuntos
Proliferação de Células/fisiologia , Dinoprosta/metabolismo , Endométrio/citologia , Células Epiteliais/fisiologia , Receptores de Prostaglandina/metabolismo , Animais , Bovinos , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Feminino , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Receptores de Prostaglandina/genética , Transdução de Sinais
17.
Oncol Res ; 28(3): 213-223, 2020 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31558185

RESUMO

PRKAA1 (protein kinase AMP-activated catalytic subunit α 1) is a catalytic subunit of AMP-activated protein kinase (AMPK), which plays a key role in regulating cellular energy metabolism through phosphorylation, and genetic variations in the PRKAA1 have been found to be associated with gastric cancer risk. However, the effect and underlying molecular mechanism of PRKAA1 on gastric cancer tumorigenesis, especially the proliferation and apoptosis, are not fully understood. Our data showed that PRKAA1 is highly expressed in BGC-823 and MKN45 cells and is expressed low in SGC-7901 and MGC-803 cells in comparison with the other gastric cancer cells. PRKAA1 downregulation by shRNA or treatment of AMPK inhibitor compound C significantly inhibited proliferation as well as promoted cell cycle arrest and apoptosis of BGC-823 and MKN45 cells. Moreover, the expression of PCNA and Bcl-2 and the activity of JNK1 and Akt signaling were also reduced in BGC-823 and MKN45 cells after PRKAA1 downregulation. In vivo experiments demonstrated that tumor growth in nude mice was significantly inhibited after PRKAA1 silencing. Importantly, inactivation of JNK1 or Akt signaling pathway significantly inhibited PRKAA1 overexpression-induced increased cell proliferation and decreased cell apoptosis in MGC-803 cells. In conclusion, our findings suggest that PRKAA1 increases proliferation and restrains apoptosis of gastric cancer cells through activating JNK1 and Akt pathways.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Apoptose , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Nus , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Neoplasias Gástricas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Cell Tissue Res ; 379(3): 577-587, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31494714

RESUMO

The current study evaluates potential applications of Sertoli cell (SC)-conditioned medium (CM) and explores the effects of the conditioned medium on the spermatogenesis process in azoospermic mice. For this study, 40 adult mice (28-30 g) were divided into 4 experimental groups: (1) control, (2) DMSO 2% (10 µl), (3) busulfan (40 mg/kg single dose) and (4) busulfan/CM (10 µl). SCs were isolated from 4-week-old mouse testes. After using anesthetics, 10 µl of CM was injected over 3-5 min into each testis and subsequently, sperm samples were collected from the tail of the epididymis. Afterward, the animals were euthanized and testis samples were taken for histopathology experiments and RNA extraction in order to examine the expression of c-kit, STRA8 and PCNA genes. The data showed that CM notably increased the total sperm count and the number of testicular cells, such as spermatogonia, primary spermatocytes, round spermatids, SCs and Leydig cells compared with the control, DMSO and busulfan groups. Furthermore, the results showed that expression of c-kit and STRA8 was significantly decreased in the busulfan and busulfan/SC groups at 8 weeks after the last injection (p < 0.001) but no significant difference was found for PCNA compared with the control and DMSO groups (p < 0.05). These findings suggest that the Sertoli cell-conditioned medium may be beneficial as a practical approach for therapeutic strategies in reproductive and regenerative medicine.


Assuntos
Células de Sertoli/citologia , Espermatogênese/fisiologia , Testículo/citologia , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose/fisiologia , Meios de Cultivo Condicionados , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Antígeno Nuclear de Célula em Proliferação/biossíntese , Antígeno Nuclear de Célula em Proliferação/genética , Proteínas Proto-Oncogênicas c-kit/biossíntese , Proteínas Proto-Oncogênicas c-kit/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células de Sertoli/metabolismo , Espermátides/citologia , Espermátides/metabolismo , Espermatócitos/citologia , Espermatócitos/metabolismo , Testículo/metabolismo
19.
Proteomics Clin Appl ; 14(1): e1800186, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31550741

RESUMO

PURPOSE: Based on the recent aptamer-related breast cancer studies, which indicate the therapeutic role of specific oligonucleotide sequences, experiments have been designed in an attempt to unravel the molecular targets of this mechanism. This article describes the study on glycoproteome changes in breast cancer cells as a result of their interactions with aptamers. EXPERIMENTAL DESIGN: Aberrations in protein glycosylation play an important role in tumorigenesis and influence cancer progression, metastasis, immunoresponse, and chemoresistance, therefore this study is focused on the identification of the alterations in glycan expression on the surface of proteins as a potential and innovative tool for biomedical applications of aptamers in cancer treatment. RESULTS: Two proteins, kinesin-like protein (KI13B) and proliferating cell nuclear antigen (PCNA), have been identified that carry N-glycan epitopes after conjugation with aptamer sequences. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple features of aptamers as an alternative to protein antibodies are utilized for various biomedical applications ranging from biomarker discovery, bioimaging, targeted therapy, drug delivery, and drug pharmacokinetics and biodistribution. Frequently, aptamers bind to their target molecules and modulate their function. Such therapeutic aptamers can modify the biological pathways for treatment of many types of diseases, such as cancer.


Assuntos
Neoplasias da Mama/genética , Cinesina/genética , Antígeno Nuclear de Célula em Proliferação/genética , Técnica de Seleção de Aptâmeros , Aptâmeros de Nucleotídeos/farmacologia , Neoplasias da Mama/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicosilação/efeitos dos fármacos , Humanos , Células MCF-7 , Polissacarídeos/genética , Proteoma/genética
20.
J Fish Biol ; 96(1): 102-110, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31674006

RESUMO

The eye of the fish has a lifelong persistent neurogenesis unlike eye of mammals, so it's highly interesting to study retinal neurogenesis and its genetic control to give complete knowledge about the cause of this property in fish in comparison to mammals. We performed fluorescent in situ hybridisation for loach Misgurnus anguillicaudatus bmi1, msi1 and sox2 genes, which are used as an indicator of the sites of multipotent stem cells. Proliferating cell nuclear antigen (PCNA), bromodeoxyuridine (BRDU) and KI67 markers were used as indicators of proliferating cells and glial fibrillary acidic protein (GFAP) immunofluorescence was used for detection of the glial property of cells, as well as, immunohistochemistry detected the role of peroxisome proliferator-activated receptor (PPAR)α and γ in retinal neurogenesis. Our results determined that the lens and the retina of loach M. anguillicaudatus contain proliferative and pluripotent stem cells that have both glial and neuroepithelial properties, which add new cells continuously throughout life even without injury-induced proliferation. The PPARα has an essential function in providing energy supply for retinal neurogenesis more than PPARγ.


Assuntos
Proliferação de Células , Cipriniformes/fisiologia , Cristalino/citologia , Retina/citologia , Células-Tronco/fisiologia , Animais , Biomarcadores , Bromodesoxiuridina/metabolismo , Cipriniformes/genética , Regulação da Expressão Gênica , Proteína Glial Fibrilar Ácida , Antígeno Ki-67/metabolismo , Cristalino/fisiologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Retina/fisiologia
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