RESUMEN
DNA damage causes genomic instability. To maintain genome integrity, cells have evolved DNA damage response, which is involved in replication fork disassembly and DNA replication termination. However, the mechanism underlying the regulation of replication fork disassembly and its connection with DNA damage repair remain elusive. The CMG-MCM7 subunit ubiquitination functions on the eukaryotic replication fork disassembly at replication termination. Until now, only ubiquitin ligases CUL2LRR1 have been reported catalyzing MCM7 ubiquitination in human cells. This study discovered that in human cells, the ubiquitin ligase RNF8 catalyzes K63-linked multi-ubiquitination of MCM7 at K145 both in vivo and in vitro. The multi-ubiquitination of MCM7 is dynamically regulated during the cell cycle, primarily presenting on chromatin during the late S phase. Additionally, MCM7 polyubiquitylation is promoted by RNF168 and BRCA1 during DNA replication termination. Upon DNA damage, the RNF8-mediated polyubiquitination of MCM7 decreased significantly during the late S phase. This study highlights the novel role of RNF8-catalyzed polyubiquitination of MCM7 in the regulation of replication fork disassembly in human cells and linking it to DNA damage response.
Asunto(s)
Daño del ADN , Replicación del ADN , Proteínas de Unión al ADN , Componente 7 del Complejo de Mantenimiento de Minicromosoma , Ubiquitina-Proteína Ligasas , Ubiquitinación , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Componente 7 del Complejo de Mantenimiento de Minicromosoma/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Células HEK293 , Reparación del ADN , Células HeLaRESUMEN
Accumulating studies suggest that splicing factors play important roles in many diseases including human cancers. Our study revealed that WBP11, a core splicing factor, is highly expressed in ovarian cancer (OC) tissues and associated with a poor prognosis. WBP11 inhibition significantly impaired the proliferation and mobility of ovarian cancer cells in vitro and in vivo. Furthermore, FOXM1 transcriptionally activated WBP11 expression by directly binding to its promoter in OC cells. Importantly, RNA-seq and alternative splicing event analysis revealed that WBP11 silencing decreased the expression of MCM7 by regulating intron 4 retention. MCM7 inhibition attenuated the increase in malignant behaviors of WBP11-overexpressing OC cells. Overall, WBP11 was identified as an oncogenic splicing factor that contributes to malignant progression by repressing intron 4 retention of MCM7 in OC cells. Thus, WBP11 is an oncogenic splicing factor with potential therapeutic and prognostic implications in OC.
Asunto(s)
Proliferación Celular , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Intrones , Componente 7 del Complejo de Mantenimiento de Minicromosoma , Neoplasias Ováricas , Humanos , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , Femenino , Intrones/genética , Componente 7 del Complejo de Mantenimiento de Minicromosoma/genética , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Animales , Línea Celular Tumoral , Ratones , Proliferación Celular/genética , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Pronóstico , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Ratones Desnudos , Empalme Alternativo/genéticaRESUMEN
Phospholipase C epsilon 1 (PLCE1) is a well-established susceptibility gene for esophageal squamous cell carcinoma (ESCC). Identification of the underlying mechanism(s) regulated by PLCE1 could lead to a better understanding of ESCC tumorigenesis. In this study, we found that PLCE1 enhances tumor progression by regulating the replicative helicase MCM7 via two pathways. PLCE1 activated PKCα-mediated phosphorylation of E2F1, which led to the transcriptional activation of MCM7 and miR-106b-5p. The increased expression of miR-106b-5p, located in intron 13 of MCM7, suppressed autophagy and apoptosis by targeting Beclin-1 and RBL2, respectively. Moreover, MCM7 cooperated with the miR-106b-25 cluster to promote PLCE1-dependent cell-cycle progression both in vivo and in vitro. In addition, PLCE1 potentiated the phosphorylation of MCM7 at six threonine residues by the atypical kinase RIOK2, which promoted MCM complex assembly, chromatin loading, and cell-cycle progression. Inhibition of PLCE1 or RIOK2 hampered MCM7-mediated DNA replication, resulting in G1-S arrest. Furthermore, MCM7 overexpression in ESCC correlated with poor patient survival. Overall, these findings provide insights into the role of PLCE1 as an oncogenic regulator, a promising prognostic biomarker, and a potential therapeutic target in ESCC. SIGNIFICANCE: PLCE1 promotes tumor progression in ESCC by activating PKCα-mediated phosphorylation of E2F1 to upregulate MCM7 and miR-106b-5p expression and by potentiating MCM7 phosphorylation by RIOK2.
Asunto(s)
Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , MicroARNs , Humanos , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/metabolismo , Fosforilación , Proteína Quinasa C-alfa/metabolismo , Línea Celular Tumoral , Fosfoinositido Fosfolipasa C/genética , Fosfoinositido Fosfolipasa C/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Regulación Neoplásica de la Expresión Génica , Proliferación Celular , Componente 7 del Complejo de Mantenimiento de Minicromosoma/genética , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismoRESUMEN
Complex cellular processes are driven by the regulated assembly and disassembly of large multiprotein complexes. While we are beginning to understand the molecular mechanism for assembly of the eukaryotic DNA replication machinery (replisome), we still know relatively little about the regulation of its disassembly at replication termination. Recently, the first elements of this process have emerged, revealing that the replicative helicase, at the heart of the replisome, is polyubiquitylated prior to unloading and that this unloading requires p97 segregase activity. Two different E3 ubiquitin ligases have now been shown to ubiquitylate the helicase under different conditions: Cul2Lrr1 and TRAIP. Here, using Xenopus laevis egg extract cell-free system and biochemical approaches, we have found two p97 cofactors, Ubxn7 and Faf1, which can interact with p97 during replisome disassembly during S-phase. We show only Ubxn7, however, facilitates efficient replisome disassembly. Ubxn7 delivers this role through its interaction via independent domains with both Cul2Lrr1 and p97 to allow coupling between Mcm7 ubiquitylation and its removal from chromatin. Our data therefore characterize Ubxn7 as the first substrate-specific p97 cofactor regulating replisome disassembly in vertebrates and a rationale for the efficacy of the Cul2Lrr1 replisome unloading pathway in unperturbed S-phase.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Cromatina , Replicación del ADN , Fase S , Proteínas de Xenopus , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Cromatina/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Ubiquitinación , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMEN
Treating yeast cells with the replication inhibitor hydroxyurea activates the S phase checkpoint kinase Rad53, eliciting responses that block DNA replication origin firing, stabilize replication forks, and prevent premature extension of the mitotic spindle. We previously found overproduction of Stn1, a subunit of the telomere-binding Cdc13-Stn1-Ten1 complex, circumvents Rad53 checkpoint functions in hydroxyurea, inducing late origin firing and premature spindle extension even though Rad53 is activated normally. Here, we show Stn1 overproduction acts through remarkably similar pathways compared to loss of RAD53, converging on the MCM complex that initiates origin firing and forms the catalytic core of the replicative DNA helicase. First, mutations affecting Mcm2 and Mcm5 block the ability of Stn1 overproduction to disrupt the S phase checkpoint. Second, loss of function stn1 mutations compensate rad53 S phase checkpoint defects. Third Stn1 overproduction suppresses a mutation in Mcm7. Fourth, stn1 mutants accumulate single-stranded DNA at non-telomeric genome locations, imposing a requirement for post-replication DNA repair. We discuss these interactions in terms of a model in which Stn1 acts as an accessory replication factor that facilitates MCM activation at ORIs and potentially also maintains MCM activity at replication forks advancing through challenging templates.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2/genética , Quinasa de Punto de Control 2/metabolismo , Replicación del ADN/genética , Componente 7 del Complejo de Mantenimiento de Minicromosoma/genética , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Mutación , Proteínas Serina-Treonina Quinasas , Fase S/genética , Puntos de Control de la Fase S del Ciclo Celular/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión a Telómeros/metabolismoRESUMEN
OBJECTIVE: Aberrant DNA replication is regarded as a component of cancer development. Minichromosome maintenance protein 7 (MCM7), which is critical for the initiation of DNA replication, is overexpressed in multiple malignancies. The effect of MCM7 on cell proliferation, apoptosis, and drug resistance of liver cancer and its mechanism were investigated in this study. METHODS: MCM7 expression in normal liver cells, liver cancer cell lines, and tissues, as well as adjacent tissues, was determined by qRT-PCR. CCK-8 and flow cytometry was performed to detect cell viability, apoptosis, and cell cycle, respectively. The related mRNA and protein expressions were detected by qRT-PCR and western blot. RESULTS: High expression of MCM7 was found in liver cancer tissues and cells, which results in notably lower survival time of patients. Cisplatin (DDP) could inhibit cell proliferation and affect MCM7 expression. Silencing of MCM7 inhibited cell viability, promoted cell apoptosis, arrested cell cycle at G1 phase, and enhanced the effect of DDP on cancer cells, while overexpression of MCM7 did the opposite. Moreover, silencing of MCM7 inhibited cyclinD1 and Ki-67 expressions. The overexpression of MCM7 increased phosphorylation levels of PI3K and AKT, activated the PI3K/AKT pathway, and weakened the inhibitory effect of DDP on the PI3K/AKT pathway. CONCLUSION: Silencing of MCM7 may inhibit cell proliferation and promote apoptosis by regulating the PI3K/AKT pathway to affect the cell cycle, thus affecting the development of liver cancer, and improving the sensitivity of liver cancer cells to DDP.
Asunto(s)
Cisplatino , Resistencia a Antineoplásicos/genética , Neoplasias Hepáticas , Componente 7 del Complejo de Mantenimiento de Minicromosoma , Transducción de Señal , Apoptosis , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Cisplatino/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Componente 7 del Complejo de Mantenimiento de Minicromosoma/genética , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
When vertebrate replisomes from neighboring origins converge, the Mcm7 subunit of the replicative helicase, CMG, is ubiquitylated by the E3 ubiquitin ligase, CRL2Lrr1. Polyubiquitylated CMG is then disassembled by the p97 ATPase, leading to replication termination. To avoid premature replisome disassembly, CRL2Lrr1 is only recruited to CMGs after they converge, but the underlying mechanism is unclear. Here, we use cryogenic electron microscopy to determine structures of recombinant Xenopus laevis CRL2Lrr1 with and without neddylation. The structures reveal that CRL2Lrr1 adopts an unusually open architecture, in which the putative substrate-recognition subunit, Lrr1, is located far from the catalytic module that catalyzes ubiquitin transfer. We further demonstrate that a predicted, flexible pleckstrin homology domain at the N-terminus of Lrr1 is essential to target CRL2Lrr1 to terminated CMGs. We propose a hypothetical model that explains how CRL2Lrr1's catalytic module is positioned next to the ubiquitylation site on Mcm7, and why CRL2Lrr1 binds CMG only after replisomes converge.
Asunto(s)
Replicación del ADN/genética , Ubiquitina-Proteína Ligasas/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Microscopía por Crioelectrón , ADN Helicasas/genética , ADN Helicasas/metabolismo , Componente 7 del Complejo de Mantenimiento de Minicromosoma/genética , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Células Sf9 , Spodoptera , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/ultraestructura , Ubiquitinación , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMEN
Uterine fibroids are the most common mesenchymal uterine neoplasms; their prevalence is estimated in 40%-60% of women under 35 and in 70%-80% of women over 50 years of age. The current research aims to focus on the etiopathogenesis of uterine fibroids, the factors that affect their growth, and markers with diagnostic and prognostic properties. The MCM (minichromosome maintenance) protein family consists of peptides whose primary function is participation in the molecular mechanism of creating replication forks while regulating DNA synthesis. The aim of this work was to determine the proliferative potential of uterine fibroid cells based on the expression of the Ki-67 antigen and the MCMs-i.e., MCM-3, MCM-5, and MCM-7. In addition, the expression of estrogen (ER) and progesterone (PgR) receptors was evaluated and correlated with the expression of the abovementioned observations. Ultimately, received results were analyzed in terms of clinical and pathological data. MATERIALS AND METHODS: In forty-four cases of uterine fibroids, immunohistochemical reactions were performed. A tissue microarray (TMA) technique was utilized and analyzed cases were assessed in triplicate. Immunohistochemistry was performed using antibodies against Ki-67 antigen, ER, PgR, MCM-3, MCM-5, and MCM-8 on an automated staining platform. Reactions were digitalized by a histologic scanner and quantified utilizing dedicated software for nuclear analysis. Assessment was based on quantification expression of the three histiospots, each representing one case in TMA. RESULTS: In the study group (uterine fibroids), statistically significant stronger expression of all the investigated MCMs was observed, as compared to the control group. In addition, moderate and strong positive correlations were found between all tested proliferative markers. The expression of the MCM-7 protein also correlated positively with ER and PgR. With regard to clinical and pathological data, there was a negative correlation between the expression of MCMs and the number of both pregnancies and births. Significant reductions in MCM-5 and MCM-7 expression were observed in the group of women receiving oral hormonal contraceptives, while smoking women showed an increase in MCM-7, ER, and PgR. CONCLUSIONS: Uterine fibroid cells have greater proliferative potential, as evaluated by expression of the Ki-67 antigen and MCMs, than unaltered myometrial cells of the uterine corpus. The expression of MCM-7 was found to have strong or moderate correlations in all assessed relations. In the context of the clinical data, as well evident proliferative potential of MCMs, further studies are strongly recommended.
Asunto(s)
Proteínas de Ciclo Celular/biosíntesis , Leiomioma/metabolismo , Componente 3 del Complejo de Mantenimiento de Minicromosoma/biosíntesis , Componente 7 del Complejo de Mantenimiento de Minicromosoma/biosíntesis , Neoplasias Uterinas/metabolismo , Adulto , Anciano , Biomarcadores de Tumor/metabolismo , Proteínas de Ciclo Celular/metabolismo , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Inmunohistoquímica , Leiomioma/patología , Persona de Mediana Edad , Componente 3 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Embarazo , Receptores de Progesterona/metabolismo , Neoplasias Uterinas/patologíaRESUMEN
Cullin ubiquitin ligases drive replisome disassembly during DNA replication termination. In worm, frog and mouse cells, CUL2LRR1 is required to ubiquitylate the MCM7 subunit of the CMG helicase. Here, we show that cullin ligases also drive CMG-MCM7 ubiquitylation in human cells, thereby making the helicase into a substrate for the p97 unfoldase. Using purified human proteins, including a panel of E2 ubiquitin-conjugating enzymes, we have reconstituted CMG helicase ubiquitylation, dependent upon neddylated CUL2LRR1. The reaction is highly specific to CMG-MCM7 and requires the LRR1 substrate targeting subunit, since replacement of LRR1 with the alternative CUL2 adaptor VHL switches ubiquitylation from CMG-MCM7 to HIF1. CUL2LRR1 firstly drives monoubiquitylation of CMG-MCM7 by the UBE2D class of E2 enzymes. Subsequently, CUL2LRR1 activates UBE2R1/R2 or UBE2G1/G2 to extend a single K48-linked ubiquitin chain on CMG-MCM7. Thereby, CUL2LRR1 converts CMG into a substrate for p97, which disassembles the ubiquitylated helicase during DNA replication termination.
Asunto(s)
Proteínas Cullin/metabolismo , ADN Helicasas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Animales , Línea Celular , Clonación Molecular/métodos , Proteínas Cullin/genética , ADN Helicasas/genética , Humanos , Immunoblotting , Lisina/metabolismo , Componente 7 del Complejo de Mantenimiento de Minicromosoma/genética , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Células Sf9 , Spodoptera , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitina-Proteína Ligasas/genética , Proteína que Contiene Valosina/genética , Proteína que Contiene Valosina/metabolismoRESUMEN
Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer that rarely develops de novo in primary tumors and is commonly acquired during the development of treatment resistance. NEPC is characterized by gain of neuroendocrine markers and loss of androgen receptor (AR), making it resistant to current therapeutic strategies targeting the AR signaling axis. Here, we report that MCM2, MCM3, MCM4, and MCM6 (MCM2/3/4/6) are elevated in human NEPC and high levels of MCM2/3/4/6 are associated with liver metastasis and poor survival in prostate cancer patients. MCM2/3/4/6 are four out of six proteins that form a core DNA helicase (MCM2-7) responsible for unwinding DNA forks during DNA replication. Inhibition of MCM2-7 by treatment with ciprofloxacin inhibits NEPC cell proliferation and migration in vitro, significantly delays NEPC tumor xenograft growth, and partially reverses the neuroendocrine phenotype in vivo. Our study reveals the clinical relevance of MCM2/3/4/6 proteins in NEPC and suggests that inhibition of MCM2-7 may represent a new therapeutic strategy for NEPC.
Asunto(s)
Carcinoma Neuroendocrino/metabolismo , Componente 2 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Tumores Neuroendocrinos/metabolismo , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células PC-3 , Receptores Androgénicos/metabolismo , Transducción de Señal/fisiología , Regulación hacia Arriba/fisiología , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Pancreatic cancer is the fourth most lethal cancer type worldwide. Due to multiple levan applications including anticancer activities, studies related to levansucrase production are of interest. To our knowledge, levan effect on pancreatic cancer cells has not been tested previously. In this work, among eighteen bacterial honey isolates, Bacillus subtilis MT453867 showed the highest levan yield (33 g/L) and levansucrase production (8.31 U/mL). One-factor-at-a-time technique increased levansucrase activity by 60% when MgSO4 was eliminated. The addition of 60 g/L banana peels enhanced the enzyme activity (192 U/mL). Placket Burman design determined the media composition for maximum levan yield (54.8 g/L) and levansucrase production (505 U/mL). The identification of levan was confirmed by thin-layer chromatography, Fourier-Transform Infrared spectrometric analysis, 13C-nuclear-magnetic resonance, and 1H-nuclear-magnetic resonance. Both crude and dialyzed levan completely inhibited the pancreatic cancer cell line at 100 ppm with no cytotoxicity on the normal retinal cell line. The LD50 of crude levan was 4833 mg/kg body weight. Levan had strong antioxidant activity and significantly reduced the expression of CXCR4 and MCM7 genes in pancreatic cancer cells with significant DNA fragmentation. In conclusion, Bacillus subtilis MT453867 levan is a promising adjunct to pancreatic-anticancer agents with both anti-cancer and chemoprotective effects.
Asunto(s)
Antineoplásicos/metabolismo , Bacillus subtilis/enzimología , Bacillus subtilis/metabolismo , Fructanos/metabolismo , Hexosiltransferasas/metabolismo , Antineoplásicos/farmacología , Fragmentación del ADN/efectos de los fármacos , Fructanos/farmacología , Humanos , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores CXCR4/metabolismo , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Minichromosome maintenance complex component 7 (MCM7) belongs to the minichromosome maintenance family that is important for the initiation of eukaryotic DNA replication. Overexpression of the MCM7 protein is relative to cellular proliferation and responsible for aggressive malignancy in various cancers. Mechanistically, inhibition of MCM7 significantly reduces the cellular proliferation associated with cancer. To date, no effective small molecular candidate has been identified that can block the progression of cancer induced by the MCM7 protein. Therefore, the study has been designed to identify small molecular-like natural drug candidates against aggressive malignancy associated with various cancers by targeting MCM7 protein. To identify potential compounds against the targeted protein a comprehensive in silico drug design including molecular docking, ADME (Absorption, Distribution, Metabolism and Excretion), toxicity, and molecular dynamics (MD) simulation approaches has been applied. Seventy phytochemicals isolated from the neem tree (Azadiractha indica) were retrieved and screened against MCM7 protein by using the molecular docking simulation method, where the top four compounds have been chosen for further evaluation based on their binding affinities. Analysis of ADME and toxicity properties reveals the efficacy and safety of the selected four compounds. To validate the stability of the protein-ligand complex structure MD simulations approach has also been performed to the protein-ligand complex structure, which confirmed the stability of the selected three compounds including CAS ID:105377-74-0, CID:12308716 and CID:10505484 to the binding site of the protein. In the study, a comprehensive data screening process has performed based on the docking, ADMET properties, and MD simulation approaches, which found a good value of the selected four compounds against the targeted MCM7 protein and indicates as a promising and effective human anticancer agent.
Asunto(s)
Azadirachta/química , Informática/métodos , Componente 7 del Complejo de Mantenimiento de Minicromosoma/antagonistas & inhibidores , Simulación de Dinámica Molecular , Neoplasias/tratamiento farmacológico , Fitoquímicos/uso terapéutico , Algoritmos , Sitios de Unión , Detección Precoz del Cáncer , Humanos , Ligandos , Componente 7 del Complejo de Mantenimiento de Minicromosoma/química , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Simulación del Acoplamiento Molecular , Terapia Molecular Dirigida/métodos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Fitoquímicos/aislamiento & purificación , Fitoquímicos/farmacología , Plantas Medicinales/química , Unión Proteica , Dominios Proteicos , TermodinámicaRESUMEN
Protein arginine methyltransferase 5 (PRMT5) is a type of methyltransferase enzyme that can catalyse arginine methylation of histones and non-histone proteins. Accumulating evidence indicates that PRMT5 promotes cancer development and progression. However, its function in colorectal cancer (CRC) is poorly understood. In this study, we revealed the oncogenic roles of PRMT5 in CRC. We found that PRMT5 promoted CRC cell proliferation, migration and invasion in vitro and in vivo. We identified minichromosome maintenance-7 (MCM7) as the direct PRMT5-binding partner. A co-immunoprecipitation (co-IP) assay indicated that PRMT5 physically interacted with MCM7 and that the direct binding domain was located between residues 1-248 in MCM7. In addition, our results from analysis of 99 CRC tissues and 77 adjacent non-cancerous tissues indicated that the PRMT5 and MCM7 expression levels were significantly higher in CRC tissues than in control tissues, which was further confirmed by bioinformatic analysis using TCGA and GEO datasets. We also found that MCM7 promoted CRC cell proliferation, migration and invasion in vitro. Furthermore, we observed that increased PRMT5 expression predicted unfavourable patient survival in CRC patients and in the subgroup of patients with a tumour size of ≤5 cm. These data suggested that PRMT5 and MCM7 might be novel potential targets for the treatment of CRC.
Asunto(s)
Carcinogénesis/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Animales , Arginina/metabolismo , Carcinogénesis/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Técnicas de Silenciamiento del Gen , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Metilación , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Unión Proteica , Dominios y Motivos de Interacción de ProteínasRESUMEN
The eukaryotic replisome is disassembled in each cell cycle, dependent upon ubiquitylation of the CMG helicase. Studies of Saccharomyces cerevisiae, Caenorhabditis elegans and Xenopus laevis have revealed surprising evolutionary diversity in the ubiquitin ligases that control CMG ubiquitylation, but regulated disassembly of the mammalian replisome has yet to be explored. Here, we describe a model system for studying the ubiquitylation and chromatin extraction of the mammalian CMG replisome, based on mouse embryonic stem cells. We show that the ubiquitin ligase CUL2LRR1 is required for ubiquitylation of the CMG-MCM7 subunit during S-phase, leading to disassembly by the p97 ATPase. Moreover, a second pathway of CMG disassembly is activated during mitosis, dependent upon the TRAIP ubiquitin ligase that is mutated in primordial dwarfism and mis-regulated in various cancers. These findings indicate that replisome disassembly in diverse metazoa is regulated by a conserved pair of ubiquitin ligases, distinct from those present in other eukaryotes.
Asunto(s)
ADN Helicasas , Replicación del ADN , Animales , Ciclo Celular/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , Ratones , Componente 7 del Complejo de Mantenimiento de Minicromosoma/genética , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Ubiquitinación , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMEN
Chemoresistance of tumors often leads to treatment failure in clinical practice, which underscores pivotal needs to uncover novel therapeutic strategies. Accumulating evidences show that microRNAs (miRNAs) are widely involved in carcinogenesis, but their function on chemoresistance remains largely unexplored. In this study, we found that miR-93-5p (miR-93) significantly inhibited cell proliferation, induced G1/S cell cycle arrest and increased chemosensitivity to paclitaxel (PTX) in vitro and in vivo. Moreover, two well-established oncogenes, E2F1 and CCND1, were identified as dual targets of miR-93. Knockdown of E2F1 and CCND1 reduced cell proliferation and PTX-sensitivity, whereas overexpression of them had the opposite effect. More importantly, overexpression of E2F1 and CCND1 antagonized miR-93-mediated cell cycle arrest and apoptosis. Further mechanistic study revealed that miR-93 exhibited its inhibitory role by directly targeting E2F1 and CCND1 to inactivate pRB/E2F1 pathway and AKT phosphorylation. Taken together, our findings suggested that miR-93 greatly improved chemosensitivity and potentially served as a novel therapeutic target for breast cancer treatment.
Asunto(s)
Neoplasias de la Mama/genética , Carcinogénesis/genética , Carcinogénesis/patología , Ciclina D1/metabolismo , Factor de Transcripción E2F1/metabolismo , MicroARNs/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Metilación de ADN/genética , Regulación hacia Abajo/genética , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Desnudos , MicroARNs/genética , Componente 7 del Complejo de Mantenimiento de Minicromosoma/genética , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Modelos Biológicos , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína de Retinoblastoma/metabolismoRESUMEN
To unravel vulnerabilities of KRAS-mutant CRC cells, a shRNA-based screen specifically inhibiting MAPK pathway components and targets was performed in CaCo2 cells harboring conditional oncogenic KRASG12V. The custom-designed shRNA library comprised 121 selected genes, which were previously identified to be strongly regulated in response to MEK inhibition. The screen showed that CaCo2 cells expressing KRASG12V were sensitive to the suppression of the DNA replication licensing factor minichromosome maintenance complex component 7 (MCM7), whereas KRASwt CaCo2 cells were largely resistant to MCM7 suppression. Similar results were obtained in an isogenic DLD-1 cell culture model. Knockdown of MCM7 in a KRAS-mutant background led to replication stress as indicated by increased nuclear RPA focalization. Further investigation showed a significant increase in mitotic cells after simultaneous MCM7 knockdown and KRASG12V expression. The increased percentage of mitotic cells coincided with strongly increased DNA damage in mitosis. Taken together, the accumulation of DNA damage in mitotic cells is due to replication stress that remained unresolved, which results in mitotic catastrophe and cell death. In summary, the data show a vulnerability of KRAS-mutant cells towards suppression of MCM7 and suggest that inhibiting DNA replication licensing might be a viable strategy to target KRAS-mutant cancers.
Asunto(s)
Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Mitosis , Mutación/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Origen de Réplica , Células CACO-2 , Muerte Celular , Proliferación Celular , Senescencia Celular , Daño del ADN , Replicación del ADN , Técnicas de Silenciamiento del Gen , Humanos , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismoRESUMEN
DNA replication is a complex process that needs to be executed accurately before cell division in order to maintain genome integrity. DNA replication is divided into three main stages: initiation, elongation and termination. One of the key events during initiation is the assembly of the replicative helicase at origins of replication, and this mechanism has been very well described over the last decades. In the last six years however, researchers have also focused on deciphering the molecular mechanisms underlying the disassembly of the replicative helicase during termination. Similar to replisome assembly, the mechanism of replisome disassembly is strictly regulated and well conserved throughout evolution, although its complexity increases in higher eukaryotes. While budding yeast rely on just one pathway for replisome disassembly in S phase, higher eukaryotes evolved an additional mitotic pathway over and above the default S phase specific pathway. Moreover, replisome disassembly has been recently found to be a key event prior to the repair of certain DNA lesions, such as under-replicated DNA in mitosis and inter-strand cross-links (ICLs) in S phase. Although replisome disassembly in human cells has not been characterised yet, they possess all of the factors involved in these pathways in model organisms, and de-regulation of many of them are known to contribute to tumorigenesis and other pathological conditions.
Asunto(s)
Replicación del ADN , Neoplasias/metabolismo , Fase S , Saccharomycetales , Animales , Caenorhabditis elegans , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Supervivencia Celular , ADN/metabolismo , Genoma , Humanos , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Mitosis , Mapeo de Interacción de Proteínas , Origen de Réplica , Saccharomyces cerevisiae , Saccharomycetales/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Xenopus laevisRESUMEN
AIMS: This study evaluates the action of Weissella paramesenteroides WpK4 on amoebic colitis. METHODS AND RESULTS: Weissella paramesenteroides WpK4 was administered in Entamoeba dispar infected and noninfected mice and clinical parameters were evaluated. Following 7 days, the caeca were collected for histopathology, morphometry and immunohistochemical staining of MUC-2, CDC-47 and IgA. The treatment reduced diarrhoea and the presence of blood in the faeces and diminished the area of necrosis, also causing weight gain. Also, the addition of this bacterium enhanced the expression of the mucin (MUC-2). The reduction in necrosis and increased CDC-47 expression indicates significant epithelial regeneration. The negative correlation between CDC-47 and the necrosis area reveals that the bacterium favoured the recovery of the necrotic regions and the positive correlation found between the expression of MUC-2 and CDC-47 indicates that the epithelial regeneration also supports the synthesis of MUC-2. CONCLUSIONS: Weissella paramesenteroides WpK4 was able to increase the protection of the intestinal mucosa against experimental amoebic colitis through the increase of MUC-2 and epithelial regeneration. SIGNIFICANCE AND IMPACT OF THE STUDY: Weissella paramesenteroides WpK4 presents the potential to become a complementary tool in the treatment of amoebic colitis.
Asunto(s)
Disentería Amebiana/prevención & control , Mucosa Intestinal/fisiología , Mucina 2/metabolismo , Regeneración , Weissella/fisiología , Animales , Modelos Animales de Enfermedad , Disentería Amebiana/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitología , Ratones , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , ProbióticosRESUMEN
High-resolution structures have not been reported for replicative helicases at a replication fork at atomic resolution, a prerequisite to understanding the unwinding mechanism. The eukaryotic replicative CMG (Cdc45, Mcm2-7, GINS) helicase contains a Mcm2-7 motor ring, with the N-tier ring in front and the C-tier motor ring behind. The N-tier ring is structurally divided into a zinc finger (ZF) sub-ring followed by the oligosaccharide/oligonucleotide-binding (OB) fold ring. Here we report the cryo-EM structure of CMG on forked DNA at 3.9 Å, revealing that parental DNA enters the ZF sub-ring and strand separation occurs at the bottom of the ZF sub-ring, where the lagging strand is blocked and diverted sideways by OB hairpin-loops of Mcm3, Mcm4, Mcm6, and Mcm7. Thus, instead of employing a specific steric exclusion process, or even a separation pin, unwinding is achieved via a "dam-and-diversion tunnel" mechanism that does not require specific protein-DNA interaction. The C-tier motor ring contains spirally configured PS1 and H2I loops of Mcms 2, 3, 5, 6 that translocate on the spirally-configured leading strand, and thereby pull the preceding DNA segment through the diversion tunnel for strand separation.
Asunto(s)
Replicación del ADN , Saccharomyces cerevisiae/enzimología , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN de Hongos/química , ADN de Hongos/genética , ADN de Hongos/metabolismo , Componente 3 del Complejo de Mantenimiento de Minicromosoma/química , Componente 3 del Complejo de Mantenimiento de Minicromosoma/genética , Componente 3 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Componente 4 del Complejo de Mantenimiento de Minicromosoma/química , Componente 4 del Complejo de Mantenimiento de Minicromosoma/genética , Componente 4 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Componente 6 del Complejo de Mantenimiento de Minicromosoma/química , Componente 6 del Complejo de Mantenimiento de Minicromosoma/genética , Componente 6 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Componente 7 del Complejo de Mantenimiento de Minicromosoma/química , Componente 7 del Complejo de Mantenimiento de Minicromosoma/genética , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Cutaneous melanoma (CM) has become a major public health concern. Studies illustrate that minichromosome maintenance protein 7 (MCM7) participate in various diseases including skin disease. Our study aimed to study the effects of MCM7 silencing on CM cell autophagy and apoptosis by modulating the AKT threonine kinase 1 (AKT1)/mechanistic target of rapamycin kinase (mTOR) signaling pathway. Initially, microarray analysis was used to screen the CM-related gene expression data as well as differentially expressed genes. Subsequently, MCM7 expression vector and lentivirus RNA used for MCM7 silencing (LV-shRNA-MCM7) were constructed, and these vectors, dimethyl sulfoxide (DMSO) and AKT activator SC79 were then introduced into CM cell line SK-MEL-2 to validate the role of MCM7 in cell autophagy, viability, apoptosis, cell cycle, migration, and invasion. To further investigate the regulatory mechanisms of MCM7 in CM progress, the expression of MCM7, AKT1, mTOR, cyclin D1, as well as autophagy and apoptosis relative factors, such as LC3B, SOD2, DJ-1, p62, Bcl-2, Bax, and caspase-3 in melanoma cells was determined. MCM7 might mediate the AKT1/mTOR signaling pathway to influence the progress of melanoma. MCM7 silencing contributed to the increased expression of Bax, capase-3, and autophagy-related genes (LC3B, SOD2, and DJ-1), but decreased the expression of Bcl-2, which suggested that MCM7 silencing promoted autophagy and cell apoptosis. At the same time, MCM7 silencing also attenuated cell viability, invasion, and migration, and reduced the cyclin D1 expression and protein levels of p-AKT1 and p-mTOR. Taken together, MCM7 silencing inhibited CM via inactivation of the AKT1/mTOR signaling pathway.