RESUMO
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.
Assuntos
Azadirachta/química , Informática/métodos , Componente 7 do Complexo de Manutenção de Minicromossomo/antagonistas & inibidores , Simulação de Dinâmica Molecular , Neoplasias/tratamento farmacológico , Compostos Fitoquímicos/uso terapêutico , Algoritmos , Sítios de Ligação , Detecção Precoce de Câncer , Humanos , Ligantes , Componente 7 do Complexo de Manutenção de Minicromossomo/química , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular/métodos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Plantas Medicinais/química , Ligação Proteica , Domínios Proteicos , TermodinâmicaRESUMO
The protein mini-chromosome maintenance 10 (Mcm10) was originally identified as an essential yeast protein in the maintenance of mini-chromosome plasmids. Subsequently, Mcm10 has been shown to be required for both initiation and elongation during chromosomal DNA replication. However, it is not fully understood how the multiple functions of Mcm10 are coordinated or how Mcm10 interacts with other factors at replication forks. Here, we identified and characterized the Mcm2-7-interacting domain in human Mcm10. The interaction with Mcm2-7 required the Mcm10 domain that contained amino acids 530-655, which overlapped with the domain required for the stable retention of Mcm10 on chromatin. Expression of truncated Mcm10 in HeLa cells depleted of endogenous Mcm10 via siRNA revealed that the Mcm10 conserved domain (amino acids 200-482) is essential for DNA replication, whereas both the conserved and the Mcm2-7-binding domains were required for its full activity. Mcm10 depletion reduced the initiation frequency of DNA replication and interfered with chromatin loading of replication protein A, DNA polymerase (Pol) α, and proliferating cell nuclear antigen, whereas the chromatin loading of Cdc45 and Pol ϵ was unaffected. These results suggest that human Mcm10 is bound to chromatin through the interaction with Mcm2-7 and is primarily involved in the initiation of DNA replication after loading of Cdc45 and Pol ϵ.
Assuntos
Cromatina/metabolismo , Replicação do DNA , Componente 2 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Origem de Replicação , Transporte Ativo do Núcleo Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Componente 2 do Complexo de Manutenção de Minicromossomo/química , Componente 7 do Complexo de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/antagonistas & inibidores , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/genética , Mutagênese Sítio-Dirigida , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Estabilidade Proteica , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Mutação Silenciosa , Homologia Estrutural de ProteínaRESUMO
Minichromosome maintenance protein (MCM7) belongs to the MCM protein family and participates in the MCM complex by playing a role in the cell replication cycle and chromosome initiation of eukaryotes. Previously, we found that several genes, including MCM7, were over-expressed in Drosophila melanogaster after white spot syndrome virus (WSSV) infection. In this study, we aimed to further research the MCM7 of kuruma shrimp, Marsupenaeus japonicus (mjMCM7) and determine its role in the innate immune system. To this end, we cloned the entire 2307-bp mjMCM7 sequence, including a 1974-bp open reading frame (ORF) encoding a 658-aa-long protein. Real-time PCR showed that the gene was primarily expressed in the hemolymph and hepatopancreas and over-expressed in shrimp challenged with WSSV. Gene function study was carried out by knocking down the expression of MCM7 using small interference RNA (siRNA). The results revealed that ß-actin, hemocyanin, prophenoloxidase (proPO) and tumor necrosis factor-α (TNF-α) were up-regulated while the cytoskeleton proteins such as myosin and Rho were significantly down-regulated at 24 h after treatment. The results indicate a possible relationship between mjMCM7 and the innate immune system, and suggest that mjMCM7 may play a role in phagocytosis. After WSSV challenge, WSSV copies and mortality count were both higher in the MCM7-siRNA-treated groups at 60 h after treatment, and the mortality count approached that of the control groups over time. The phagocytosis rate was significantly lower in the MCM7-siRNA-treated group than in the WSSV group. The findings of this study confirm that mjMCM7 positively regulates phagocytosis and plays an important role against WSSV. These results could help researchers to further understand the function of the MCM7 protein and reveal its potential role in the innate immunity of invertebrates.
Assuntos
Proteínas de Artrópodes/genética , Imunidade Inata/genética , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Penaeidae/genética , Penaeidae/imunologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Componente 7 do Complexo de Manutenção de Minicromossomo/química , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Análise de Sequência com Séries de Oligonucleotídeos , Penaeidae/metabolismo , Penaeidae/virologia , Fagocitose , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de SequênciaRESUMO
The replicative mini-chromosome-maintenance 2-7 (MCM2-7) helicase is loaded in Saccharomyces cerevisiae and other eukaryotes as a head-to-head double-hexamer around origin DNA. At first, ORC/Cdc6 recruits with the help of Cdt1 a single MCM2-7 hexamer to form an 'initial' ORC/Cdc6/Cdt1/MCM2-7 complex. Then, on ATP hydrolysis and Cdt1 release, the 'initial' complex is transformed into an ORC/Cdc6/MCM2-7 (OCM) complex. However, it remains unclear how the OCM is subsequently converted into a MCM2-7 double-hexamer. Through analysis of MCM2-7 hexamer-interface mutants we discovered a complex competent for MCM2-7 dimerization. We demonstrate that these MCM2-7 mutants arrest during prereplicative complex (pre-RC) assembly after OCM formation, but before MCM2-7 double-hexamer assembly. Remarkably, only the OCM complex, but not the 'initial' ORC/Cdc6/Cdt1/MCM2-7 complex, is competent for MCM2-7 dimerization. The MCM2-7 dimer, in contrast to the MCM2-7 double-hexamer, interacts with ORC/Cdc6 and is salt-sensitive, classifying the arrested complex as a helicase-loading intermediate. Accordingly, we found that overexpression of the mutants cause cell-cycle arrest and dominant lethality. Our work identifies the OCM complex as competent for MCM2-7 dimerization, reveals MCM2-7 dimerization as a limiting step during pre-RC formation and defines critical mechanisms that explain how origins are licensed.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Pontos de Checagem do Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Componente 7 do Complexo de Manutenção de Minicromossomo/química , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Dados de Sequência Molecular , Mutação , Multimerização Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The MCM2-7 helicase is a heterohexameric complex with essential roles as part of both the pre-replication and pre-initiation complexes in the early stages of DNA replication. Meier-Gorlin syndrome, a rare primordial dwarfism, is strongly associated with disruption to the pre-replication complex, including a single case described with variants in MCM5. Conversely, a biallelic pathogenic variant in MCM4 underlies immune deficiency with growth retardation, features also seen in individuals with pathogenic variants in other pre-initiation complex encoding genes such as GINS1, MCM10, and POLE. Through exome and chromium genome sequencing, supported by functional studies, we identify biallelic pathogenic variants in MCM7 and a strong candidate biallelic pathogenic variant in MCM3. We confirm variants in MCM7 are deleterious and through interfering with MCM complex formation, impact efficiency of S phase progression. The associated phenotypes are striking; one patient has typical Meier-Gorlin syndrome, whereas the second case has a multi-system disorder with neonatal progeroid appearance, lipodystrophy and adrenal insufficiency. We provide further insight into the developmental complexity of disrupted MCM function, highlighted by two patients with a similar variant profile in MCM7 but disparate clinical features. Our results build on other genetic findings linked to disruption of the pre-replication and pre-initiation complexes, and the replisome, and expand the complex clinical genetics landscape emerging due to disruption of DNA replication.
Assuntos
Insuficiência Adrenal/diagnóstico , Insuficiência Adrenal/genética , Microtia Congênita/diagnóstico , Microtia Congênita/genética , Transtornos do Crescimento/diagnóstico , Transtornos do Crescimento/genética , Lipodistrofia/diagnóstico , Lipodistrofia/genética , Micrognatismo/diagnóstico , Micrognatismo/genética , Componente 3 do Complexo de Manutenção de Minicromossomo/genética , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Patela/anormalidades , Adolescente , Alelos , Sequência de Aminoácidos , Ciclo Celular/genética , Criança , Pré-Escolar , Fácies , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Genótipo , Humanos , Lactente , Masculino , Componente 3 do Complexo de Manutenção de Minicromossomo/química , Componente 7 do Complexo de Manutenção de Minicromossomo/química , Modelos Moleculares , Nova Zelândia , Fenótipo , Conformação ProteicaRESUMO
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.
Assuntos
Replicação do DNA , Saccharomyces cerevisiae/enzimologia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , Componente 3 do Complexo de Manutenção de Minicromossomo/química , Componente 3 do Complexo de Manutenção de Minicromossomo/genética , Componente 3 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 4 do Complexo de Manutenção de Minicromossomo/química , Componente 4 do Complexo de Manutenção de Minicromossomo/genética , Componente 4 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 6 do Complexo de Manutenção de Minicromossomo/química , Componente 6 do Complexo de Manutenção de Minicromossomo/genética , Componente 6 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 7 do Complexo de Manutenção de Minicromossomo/química , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The amino-terminal region of eukaryotic MCM4 is characteristic of the presence of a number of phosphorylation sites for CDK and DDK, suggesting that the region plays regulatory roles in the MCM2-7 helicase function. However, the roles are not fully understood. We analyzed the role of the amino-terminal region of human MCM4 by using MCM4/6/7 helicase as a model for MCM2-7 helicase. First we found that deletion of 35 amino acids at the amino-terminal end resulted in inhibition of DNA helicase activity of the MCM4/6/7 complex. Conversion of arginine at amino acid no. 10 and 11 to alanine had similar effect to the deletion mutant of Δ1-35, suggesting that these arginine play a role in the DNA helicase activity. The data suggest that expression of these mutant MCM4 in HeLa cells perturbed the progression of the S phase. Substitution of six CDK phosphorylation sites (3, 7, 19, 32, 54 and 110) in the amino-terminal region by phospho-mimetic glutamic acids affected the hexamer formation of the MCM4/6/7 complex. MCM4 phosphorylation by CDK may play a role in DNA replication licensing system, and the present results suggest that the phosphorylation interferes MCM function by lowering stability of MCM complex.
Assuntos
Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Componente 4 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 6 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Processamento de Proteína Pós-Traducional , Fase S , Substituição de Aminoácidos , Arginina/química , Estabilidade Enzimática , Deleção de Genes , Células HeLa , Humanos , Componente 4 do Complexo de Manutenção de Minicromossomo/química , Componente 4 do Complexo de Manutenção de Minicromossomo/genética , Componente 6 do Complexo de Manutenção de Minicromossomo/química , Componente 6 do Complexo de Manutenção de Minicromossomo/genética , Componente 7 do Complexo de Manutenção de Minicromossomo/química , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Mutagênese Sítio-Dirigida , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação , Mutação Puntual , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
An MCM4 mutation detected in human cancer cells from endometrium was characterized. The mutation of G486D is located within MCM-box and the glycine at 486 in human MCM4 is conserved in Saccharomyces cerevisiae MCM4 and Sulfolobus solfataricus MCM. This MCM4 mutation affected human MCM4/6/7 complex formation, since the complex containing the mutant MCM4 protein is unstable and the mutant MCM4 protein is tend to be degraded. It is likely that the MCM4 mutation affects the interaction with MCM7 to destabilize the MCM4/6/7 complex. Cells with abnormal nuclear morphology were detected when the mutant MCM4 was expressed in HeLa cells, suggesting that DNA replication was perturbed in the presence of the mutant MCM4. Role of the conserved amino acid in MCM4 function is discussed.
Assuntos
Componente 4 do Complexo de Manutenção de Minicromossomo/genética , Componente 4 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 6 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteínas Mutantes/metabolismo , Mutação , Células HeLa , Humanos , Componente 4 do Complexo de Manutenção de Minicromossomo/química , Componente 6 do Complexo de Manutenção de Minicromossomo/química , Componente 6 do Complexo de Manutenção de Minicromossomo/genética , Componente 7 do Complexo de Manutenção de Minicromossomo/química , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Complexos Multiproteicos/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Estabilidade ProteicaRESUMO
Nuclear DNA duplication in the absence of cell division (i.e. endoreplication) leads to somatic polyploidy in eukaryotic cells. In contrast to some invertebrate neurons, whose nuclei may contain up to 200,000-fold the normal haploid DNA amount (C), polyploid neurons in higher vertebrates show only 4C DNA content. To explore the mechanism that prevents extra rounds of DNA synthesis in these latter cells we focused on the chick retina, where a population of tetraploid retinal ganglion cells (RGCs) has been described. We show that differentiating chick RGCs that express the neurotrophic receptors p75 and TrkB while lacking retinoblastoma protein, a feature of tetraploid RGCs, also express p27(Kip1). Two different short hairpin RNAs (shRNA) that significantly downregulate p27(Kip1) expression facilitated DNA synthesis and increased ploidy in isolated chick RGCs. Moreover, this forced DNA synthesis could not be prevented by Cdk4/6 inhibition, thus suggesting that it is triggered by a mechanism similar to endoreplication. In contrast, p27(Kip1) deficiency in mouse RGCs does not lead to increased ploidy despite previous observations have shown ectopic DNA synthesis in RGCs from p27(Kip1-/-) mice. This suggests that a differential mechanism is used for the regulation of neuronal endoreplication in mammalian versus avian RGCs.