RESUMO
Cyclin-dependent kinase 7 (Cdk7) is required in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of transcription factor TFIIH. Cdk7 forms active complexes by associating with Cyclin H and Mat1, and is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates basic residues conserved in other CDKs, pS164 nucleates an arginine network unique to the ternary Cdk7 complex, involving all three subunits. We identify differential dependencies of kinase activity and substrate recognition on the individual phosphorylations. CAK function is unaffected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by T170 phosphorylation. Moreover, dual T-loop phosphorylation stimulates multisite phosphorylation of the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7 activation is a two-step process wherein S164 phosphorylation precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing processivity, while pT170 enhances activity towards key transcriptional substrates.
Assuntos
Quinase Ativadora de Quinase Dependente de Ciclina , Quinases Ciclina-Dependentes , Fosforilação , Humanos , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Ciclina H/metabolismo , Ciclina H/química , Ciclina H/genética , Cristalografia por Raios X , RNA Polimerase II/metabolismo , RNA Polimerase II/química , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/genética , Modelos Moleculares , Fatores de Transcrição/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Domínios Proteicos , Proteínas de Ciclo CelularRESUMO
The infection of human cytomegalovirus (HCMV) is strongly determined by the host-cell interaction in a way that the efficiency of HCMV lytic replication is dependent on the regulatory interplay between viral and cellular proteins. In particular, the activities of protein kinases, such as cyclin-dependent kinases (CDKs) and the viral CDK ortholog (vCDK/pUL97), play an important role in both viral reproduction and virus-host interaction. Very recently, we reported on the complexes formed between vCDK/pUL97, human cyclin H, and CDK7. Major hallmarks of this interplay are the interaction between cyclin H and vCDK/pUL97, which is consistently detectable across various conditions and host cell types of infection, the decrease or increase in pUL97 kinase activity resulting from cyclin H knock-down or elevated levels, respectively, and significant trans-stimulation of human CDK7 activity by pUL97 in vitro. Due to the fact that even a ternary complex of vCDK/pUL97-cyclin H-CDK7 can be detected by coimmunoprecipitation and visualized by bioinformatic structural modeling, we postulated a putative impact of the respective kinase activities on the patterns of transcription in HCMV-infected cells. Here, we undertook a first vCDK/pUL97-specific transcriptomic analysis, which combined conditions of fully lytic HCMV replication with those under specific vCDK/pUL97 or CDK7 drug-mediated inhibition or transient cyclin H knockout. The novel results were further strengthened using bioinformatic modeling of the involved multi-protein complexes. Our data underline the importance of these kinase activities for the C-terminal domain (CTD) phosphorylation-driven activation of host RNA polymerase in HCMV-infected cells. The impact of the individual experimental conditions on differentially expressed gene profiles is described in detail and discussed.
Assuntos
Ciclinas , Infecções por Herpesviridae , Humanos , Ciclinas/metabolismo , Citomegalovirus/genética , Ciclina H/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , FosforilaçãoRESUMO
The complex host interaction network of human cytomegalovirus (HCMV) involves the regulatory protein kinase pUL97, which represents a viral cyclin-dependent kinase (CDK) ortholog. pUL97 interacts with the three human cyclin types T1, H, and B1, whereby the binding region of cyclin T1 and the pUL97 oligomerization region were both assigned to amino acids 231-280. We further addressed the question of whether HCMVs harboring mutations in ORF-UL97, i.e., short deletions or resistance-conferring point mutations, are affected in the interaction with human cyclins and viral replication. To this end, clinically relevant UL97 drug-resistance-conferring mutants were analyzed by whole-genome sequencing and used for genetic marker transfer experiments. The recombinant HCMVs indicated conservation of pUL97-cyclin interaction, since all viral UL97 point mutants continued to interact with the analyzed cyclin types and exerted wild-type-like replication fitness. In comparison, recombinant HCMVs UL97 Δ231-280 and also the smaller deletion Δ236-275, but not Δ241-270, lost interaction with cyclins T1 and H, showed impaired replication efficiency, and also exhibited reduced kinase activity. Moreover, a cellular knock-out of cyclins B1 or T1 did not alter HCMV replication phenotypes or pUL97 kinase activity, possibly indicating alternative, compensatory pUL97-cyclin interactions. In contrast, however, cyclin H knock-out, similar to virus deletion mutants in the pUL97-cyclin H binding region, exhibited strong defective phenotypes of HCMV replication, as supported by reduced pUL97 kinase activity in a cyclin H-dependent coexpression setting. Thus, cyclin H proved to be a very relevant determinant of pUL97 kinase activity and viral replication efficiency. As a conclusion, the results provide evidence for the functional importance of pUL97-cyclin interaction. High selective pressure on the formation of pUL97-cyclin complexes was identified by the use of clinically relevant mutants.
Assuntos
Ciclina H , Citomegalovirus , Proteínas Virais , Aminoácidos/metabolismo , Ciclina H/genética , Ciclina H/metabolismo , Ciclina T/genética , Ciclina T/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Citomegalovirus/fisiologia , Marcadores Genéticos , Humanos , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Virais/genética , Replicação Viral/genéticaRESUMO
INTRODUCTION: Studies have previously shown that Cyclin H (CCNH) is involved in the tumorigenesis and development of many cancers. The increasing research in CCNH is associated with the poor prognosis of most human cancers. Early diagnosis and clinical treatment are still the main challenges for lung cancer treatment. However, the exact role of CCNH in the tumorigenesis of lung cancer remains unclear. METHODS: The Tumor Genome Atlas (TCGA) database and the Clinical Proteomics Tumor Analysis Association (CPTAC) database were analyzed to detect key genes that might play an important role in lung cancer. The biological functions of CCNH were further revealed through bioinformatics experiments. The Kaplan-Meier method was applied to explore the relationship between CCNH expression and prognosis. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the expression levels of CCNH in 6 lung cancer tissues and 3 cancer cell lines. The effect of CCNH expression on lung cancer progression was studied by in vitro functional experiments. RESULTS: Database analysis screened out candidate oncogenes, and CCNH was of great significance to the tumorigenesis of lung cancer. The higher the expression of CCNH was, the lower the survival rate of lung cancer patients were. The qRT-PCR data illustrated that the CCNH expression level was largely increased in lung cancer tissues and cells. The reduction of CCNH inhibited cell proliferation, invasion, and migration. CONCLUSION: CCNH was related to poor prognosis, suggesting that CCNH regulated the tumorigenesis and development of lung cancer. Our study suggested that CCNH was a promising biomarker and target in the treatment of lung cancer.
Assuntos
Ciclina H/genética , Neoplasias Pulmonares/genética , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Biomarcadores Tumorais/genética , Carcinogênese/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Biologia Computacional , Ciclina H/metabolismo , Bases de Dados Genéticas/estatística & dados numéricos , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Prognóstico , Mapas de Interação de Proteínas , ProteômicaRESUMO
Cyclin-dependent kinase 7 (CDK7), Cyclin H, and the RING-finger protein MAT1 form the heterotrimeric CDK-activating kinase (CAK) complex which is vital for transcription and cell-cycle control. When associated with the general transcription factor II H (TFIIH) it activates RNA polymerase II by hyperphosphorylation of its C-terminal domain (CTD). In the absence of TFIIH the trimeric complex phosphorylates the T-loop of CDKs that control cell-cycle progression. CAK holds a special position among the CDK branch due to this dual activity and the dependence on two proteins for activation. We solved the structure of the CAK complex from the model organism Chaetomium thermophilum at 2.6-Å resolution. Our structure reveals an intricate network of interactions between CDK7 and its two binding partners MAT1 and Cyclin H, providing a structural basis for the mechanism of CDK7 activation and CAK activity regulation. In vitro activity measurements and functional mutagenesis show that CDK7 activation can occur independent of T-loop phosphorylation and is thus exclusively MAT1-dependent by positioning the CDK7 T-loop in its active conformation.
Assuntos
Ciclina H , Quinases Ciclina-Dependentes , Ciclo Celular , Chaetomium/química , Chaetomium/enzimologia , Ciclina H/química , Ciclina H/metabolismo , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fosforilação , Transcrição Gênica , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Human cytomegalovirus (HCMV) is a common ß-herpesvirus causing life-long latent infections. HCMV replication interferes with cell cycle regulation in host cells because the HCMV-encoded cyclin-dependent kinase (CDK) ortholog pUL97 extensively phosphorylates the checkpoint regulator retinoblastoma protein. pUL97 also interacts with cyclins B1, T1, and H, and recent findings have strongly suggested that these interactions influence pUL97 substrate recognition. Interestingly, here we detected profound mechanistic differences among these pUL97-cyclin interactions. Our study revealed the following. (i) pUL97 interacts with cyclins B1 and H in a manner dependent on pUL97 activity and HCMV-specific cyclin modulation, respectively. (ii) The phosphorylated state of both proteins is an important determinant of the pUL97-cyclin B1 interaction. (iii) Activated phospho-Thr-315 cyclin H is up-regulated during HCMV replication. (iv) Thr-315 phosphorylation is independent of intracellular pUL97 or CDK7 activity. (v) pUL97-mediated in vitro phosphorylation is detectable for cyclin B1 but not H. (vi) Mutual transphosphorylation between pUL97 and CDK7 is not detectable, and an MS-based phosphosite analysis indicated that pUL97 might unexpectedly not be phosphorylated in its T-loop. (vii) The binary complexes pUL97-cyclin H and CDK7-cyclin H as well as the ternary complex pUL97-cyclin-H-CDK7 are detectable in an assembly-based CoIP approach. (viii) pUL97 self-interaction can be bridged by the transcriptional cyclins T1 or H but not by the classical cell cycle-regulating B1 cyclin. Combined, our findings unravel a number of cyclin type-specific differences in pUL97 interactions and suggest a multifaceted regulatory impact of cyclins on HCMV replication.
Assuntos
Ciclina B1/metabolismo , Ciclina H/metabolismo , Ciclina T/metabolismo , Citomegalovirus/fisiologia , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Ciclina B1/genética , Ciclina H/genética , Ciclina T/genética , Células HEK293 , Humanos , Fosforilação , Domínios Proteicos , Estrutura Quaternária de Proteína , Proteínas Virais/genéticaRESUMO
The avian reovirus p17 protein is a nucleocytoplasmic shuttling protein. Although we have demonstrated that p17 causes cell growth retardation via activation of p53, the precise mechanisms remain unclear. This is the first report that avian reovirus p17 possesses broad inhibitory effects on cell cycle CDKs, cyclins, CDK-cyclin complexes, and CDK-activating kinase activity in various mammalian, avian, and cancer cell lines. Suppression of CDK activity by p17 occurs by direct binding to CDKs, cyclins, and CDK-cyclin complexes; transcriptional down-regulation of CDKs; cytoplasmic retention of CDKs and cyclins; and inhibition of CDK-activating kinase activity by promoting p53-cyclin H interaction. p17 binds to CDK-cyclin except for CDK1-cyclin B1 and CDK7-cyclin H complexes. We have determined that the negatively charged 151LAVXDVDA(E/D)DGADPN165 motif in cyclin B1 interacts with a positively charged region of CDK1. p17 mimics the cyclin B1 sequence to compete for CDK1 binding. The PSTAIRE motif is not required for interaction of CDK1-cyclin B1, but it is required for other CDK-cyclin complexes. p17 interacts with cyclins by its cyclin-binding motif, 125RXL127 Sequence and mutagenic analyses of p17 indicated that a 140WXFD143 motif and residues Asp-113 and Lys-122 in p17 are critical for CDK2 and CDK6 binding, leading to their sequestration in the cytoplasm. Exogenous expression of p17 significantly enhanced virus replication, whereas p17 mutants with low binding ability to cell cycle CDKs had no effect on virus yield, suggesting that p17 inhibits cell growth and the cell cycle, benefiting virus replication. An in vivo tumorigenesis assay also showed a significant reduction in tumor size.
Assuntos
Ciclina H/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Orthoreovirus Aviário/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Virais/metabolismo , Animais , Ciclo Celular , Embrião de Galinha , Chlorocebus aethiops , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ciclina H/genética , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/antagonistas & inibidores , Humanos , Infecções por Reoviridae/virologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Células Vero , Proteínas Virais/genéticaRESUMO
BACKGROUND/AIM: The aim of the present study was to investigate the radio-sensitizing efficacy of curcumin, a traditional Chinese medicine (TCM) on colon cancer cells in vitro and in vivo. MATERIALS AND METHODS: Human colon cancer HT-29 cells were treated with curcumin (2.5 µM), irradiation (10 Gy) and the combination of irradiation and curcumin. Cell proliferation was assessed using the MTT assay. Apoptotic cells were detected by Annexin V-PE/7-AAD analysis. PCR was performed to determine differential-expression profiling of 95 DNA-repair genes in irradiated cells and cells treated with both irradiation and curcumin. Differentially-expressed genes were confirmed by Western blotting. In vivo radio-sensitizing efficacy of curcumin was assessed in a xenograft mouse model of HT-29 colon cancer. Curcumin was administrated daily by intraperitoneal injection at 20 mg/kg/dose. Mice received irradiation (10 Gy) twice weekly. Apoptosis of the cancer cells following treatment was determined by TUNEL staining. RESULTS: Irradiation induced proliferation inhibition and apoptosis of HT-29 cells in vitro. Concurrent curcumin treatment sensitized the HT-29 tumor to irradiation (p<0.01). DNA repair-related genes CCNH and XRCC5 were upregulated and LIG4 and PNKP downregulated by the combination of curcumin and irradiation compared with irradiation alone (p<0.05). Combined treatment of curcumin and irradiation resulted in a significantly greater tumor-growth inhibition and apoptosis compared to irradiation treatment alone (p<0.01). CONCLUSION: Curcumin sensitizes human colon cancer in vitro and in vivo to radiation. Downregulation of LIG4 and PNKP and upregulation of XRCC5 and CCNH DNA-repair-related genes were involved in the radio-sensitizing efficacy of curcumin in colon cancer.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/radioterapia , Curcumina/farmacologia , Curcumina/uso terapêutico , Radiossensibilizantes/farmacologia , Radiossensibilizantes/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Ciclina H/genética , Ciclina H/metabolismo , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Células HT29 , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Medicina Tradicional Chinesa , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Carga Tumoral/efeitos dos fármacosRESUMO
BACKGROUND: Genomic analysis of drug response can provide unique insights into therapies that can be used to match the "right drug to the right patient." However, the process of discovering such therapeutic insights using genomic data is not straightforward and represents an area of active investigation. EDDY (Evaluation of Differential DependencY), a statistical test to detect differential statistical dependencies, is one method that leverages genomic data to identify differential genetic dependencies. EDDY has been used in conjunction with the Cancer Therapeutics Response Portal (CTRP), a dataset with drug-response measurements for more than 400 small molecules, and RNAseq data of cell lines in the Cancer Cell Line Encyclopedia (CCLE) to find potential drug-mediator pairs. Mediators were identified as genes that showed significant change in genetic statistical dependencies within annotated pathways between drug sensitive and drug non-sensitive cell lines, and the results are presented as a public web-portal (EDDY-CTRP). However, the interpretability of drug-mediator pairs currently hinders further exploration of these potentially valuable results. METHODS: In this study, we address this challenge by constructing evidence networks built with protein and drug interactions from the STITCH and STRING interaction databases. STITCH and STRING are sister databases that catalog known and predicted drug-protein interactions and protein-protein interactions, respectively. Using these two databases, we have developed a method to construct evidence networks to "explain" the relation between a drug and a mediator. RESULTS: We applied this approach to drug-mediator relations discovered in EDDY-CTRP analysis and identified evidence networks for ~70% of drug-mediator pairs where most mediators were not known direct targets for the drug. Constructed evidence networks enable researchers to contextualize the drug-mediator pair with current research and knowledge. Using evidence networks, we were able to improve the interpretability of the EDDY-CTRP results by linking the drugs and mediators with genes associated with both the drug and the mediator. CONCLUSION: We anticipate that these evidence networks will help inform EDDY-CTRP results and enhance the generation of important insights to drug sensitivity that will lead to improved precision medicine applications.
Assuntos
Preparações Farmacêuticas/metabolismo , Proteínas/metabolismo , Linhagem Celular , Ciclina H/química , Ciclina H/genética , Ciclina H/metabolismo , Reparo do DNA , Bases de Dados Factuais , Proteínas Quinases Associadas com Morte Celular/química , Proteínas Quinases Associadas com Morte Celular/genética , Proteínas Quinases Associadas com Morte Celular/metabolismo , Redes Reguladoras de Genes , Humanos , Imidazóis/química , Imidazóis/metabolismo , Preparações Farmacêuticas/química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/química , Proteínas/genética , Triazinas/química , Triazinas/metabolismoRESUMO
All well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs), and these protein kinase complexes are viable drug targets. The regulatory control of the Plasmodium falciparum cell division cycle remains poorly understood, and the roles of the various CDKs and cyclins remain unclear. The P. falciparum genome contains multiple CDKs, but surprisingly, it does not contain any sequence-identifiable G1-, S-, or M-phase cyclins. We demonstrate that P. falciparum Cyc1 (PfCyc1) complements a G1 cyclin-depleted Saccharomyces cerevisiae strain and confirm that other identified malaria parasite cyclins do not complement this strain. PfCyc1, which has the highest sequence similarity to the conserved cyclin H, cannot complement a temperature-sensitive yeast cyclin H mutant. Coimmunoprecipitation of PfCyc1 from P. falciparum parasites identifies PfMAT1 and PfMRK as specific interaction partners and does not identify PfPK5 or other CDKs. We then generate an endogenous conditional allele of PfCyc1 in blood-stage P. falciparum using a destabilization domain (DD) approach and find that PfCyc1 is essential for blood-stage proliferation. PfCyc1 knockdown does not impede nuclear division, but it prevents proper cytokinesis. Thus, we demonstrate that PfCyc1 has a functional divergence from bioinformatic predictions, suggesting that the malaria parasite cell division cycle has evolved to use evolutionarily conserved proteins in functionally novel ways.IMPORTANCE Human infection by the eukaryotic parasite Plasmodium falciparum causes malaria. Most well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs) to promote essential cell division processes. Remarkably, there are no identifiable cyclins that are predicted to control the cell cycle in the malaria parasite genome. Thus, our knowledge regarding the basic mechanisms of the malaria parasite cell cycle remains unsatisfactory. We demonstrate that P. falciparum Cyc1 (PfCyc1), a transcriptional cyclin homolog, complements a cell cycle cyclin-deficient yeast strain but not a transcriptional cyclin-deficient strain. We show that PfCyc1 forms a complex in the parasite with PfMRK and the P. falciparum MAT1 homolog. PfCyc1 is essential and nonredundant in blood-stage P. falciparum PfCyc1 knockdown causes a stage-specific arrest after nuclear division, demonstrating morphologically aberrant cytokinesis. This work demonstrates a conserved PfCyc1/PfMAT1/PfMRK complex in malaria and suggests that it functions as a schizont stage-specific regulator of the P. falciparum life cycle.
Assuntos
Ciclina H/metabolismo , Citocinese , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Proteínas de Protozoários/metabolismo , Ciclo Celular/genética , Ciclina H/química , Ciclina H/genética , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Citocromos c/genética , Citocromos c/metabolismo , Citocinese/genética , Estágios do Ciclo de Vida/genética , Mutação , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/genética , Reprodução Assexuada/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Cigarette smoke derivatives like NNK (4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone) and NNAL (4-(methylnitrosamino)-1-(3-pyridyl)-1-butan-1-ol) are well-known carcinogens. We analyzed the interaction of enzymes involved in the NER (nucleotide excision repair) pathway with ligands (NNK and NNAL). Binding was characterized for the enzymes sharing equivalent or better interaction as compared to +Ve control. The highest obtained docking energy between NNK and enzymes RAD23A, CCNH, CDK7, and CETN2 were -7.13 kcal/mol, -7.27 kcal/mol, -8.05 kcal/mol and -7.58 kcal/mol respectively. Similarly the highest obtained docking energy between NNAL and enzymes RAD23A, CCNH, CDK7, and CETN2 were -7.46 kcal/mol, -7.94 kcal/mol, -7.83 kcal/mol and -7.67 kcal/mol respectively. In order to find out the effect of NNK and NNAL on enzymes involved in the NER pathway applying protein-protein interaction and protein-complex (i.e. enzymes docked with NNK/NNAL) interaction analysis. It was found that carcinogens are well capable to reduce the normal functioning of genes like RAD23A (HR23A), CCNH, CDK7 and CETN2. In silico analysis indicated loss of functions of these genes and their corresponding enzymes, which possibly might be a cause for alteration of DNA repair pathways leading to damage buildup and finally contributing to cancer formation.
Assuntos
Algoritmos , Proteínas de Ligação ao Cálcio/metabolismo , Carcinógenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina H/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Nitrosaminas/metabolismo , Piridinas/metabolismo , Proteínas de Ligação ao Cálcio/química , Carcinógenos/química , Proteínas de Ciclo Celular/química , Cromatografia Líquida de Alta Pressão , Biologia Computacional/métodos , Ciclina H/química , Quinases Ciclina-Dependentes/química , Reparo do DNA , Enzimas Reparadoras do DNA/química , Proteínas de Ligação a DNA/química , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Nitrosaminas/química , Domínios e Motivos de Interação entre Proteínas , Piridinas/química , Fumar , Quinase Ativadora de Quinase Dependente de CiclinaAssuntos
Doenças das Artérias Carótidas/enzimologia , Proliferação de Células , Ciclina H/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Células Endoteliais/enzimologia , Células Endoteliais da Veia Umbilical Humana/enzimologia , MicroRNAs/metabolismo , Transcrição Gênica , Animais , Humanos , MasculinoRESUMO
OBJECTIVE: The site-specificity of endothelial phenotype is attributable to the local hemodynamic forces. The flow regulation of microRNAs in endothelial cells (ECs) plays a significant role in vascular homeostasis and diseases. The objective of this study was to elucidate the molecular mechanism by which the pulsatile shear flow-induced microRNA-23b (miR-23b) exerts antiproliferative effects on ECs. APPROACH AND RESULTS: We used a combination of a cell perfusion system and experimental animals to examine the flow regulation of miR-23b in modulating EC proliferation. Our results demonstrated that pulsatile shear flow induces the transcription factor Krüppel-like factor 2 to promote miR-23b biosynthesis; the increase in miR-23b then represses cyclin H to impair the activity and integrity of cyclin-dependent kinase-activating kinase (CAK) complex. The inhibitory effect of miR-23b on CAK exerts dual actions to suppress cell cycle progression, and reduce basal transcription by deactivating RNA polymerase II. Whereas pulsatile shear flow regulates the miR-23b/CAK pathway to exert antiproliferative effects on ECs, oscillatory shear flow has little effect on the miR-23b/CAK pathway and hence does not cause EC growth arrest. Such flow pattern-dependent phenomena are validated with an in vivo model on rat carotid artery: the flow disturbance induced by partial carotid ligation led to a lower expression of miR-23b and a higher EC proliferation in comparison with the pulsatile flow regions of the unligated vessels. Local delivery of miR-23b mitigated the proliferative EC phenotype in partially ligated vessels. CONCLUSIONS: Our findings unveil a novel mechanism by which hemodynamic forces modulate EC proliferative phenotype through the miR-23b/CAK pathway.
Assuntos
Doenças das Artérias Carótidas/enzimologia , Proliferação de Células , Ciclina H/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Células Endoteliais/enzimologia , Células Endoteliais da Veia Umbilical Humana/enzimologia , MicroRNAs/metabolismo , Transcrição Gênica , Animais , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/fisiopatologia , Pontos de Checagem do Ciclo Celular , Células Cultivadas , Ciclina H/genética , Quinases Ciclina-Dependentes/genética , Modelos Animais de Doenças , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Mecanotransdução Celular , MicroRNAs/genética , Perfusão , Fenótipo , Fluxo Pulsátil , Interferência de RNA , RNA Polimerase II/metabolismo , Ratos , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional , Estresse Mecânico , Fatores de Tempo , Transfecção , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Arabidopsis (Arabidopsis thaliana) CYCLIN-DEPENDENT KINASE Ds (CDKDs) phosphorylate the C-terminal domain of the largest subunit of RNA polymerase II. Arabidopsis CYCLIN H;1 (CYCH;1) interacts with and activates CDKDs; however, the physiological function of CYCH;1 has not been determined. Here, we report that CYCH;1, which is localized to the nucleus, positively regulates blue light-induced stomatal opening. Reduced-function cych;1 RNA interference (cych;1 RNAi) plants exhibited a drought tolerance phenotype. CYCH;1 is predominantly expressed in guard cells, and its expression was substantially down-regulated by dehydration. Transpiration of intact leaves was reduced in cych;1 RNAi plants compared with the wild-type control in light but not in darkness. CYCH;1 down-regulation impaired blue light-induced stomatal opening but did not affect guard cell development or abscisic acid-mediated stomatal closure. Microarray and real-time polymerase chain reaction analyses indicated that CYCH;1 did not regulate the expression of abscisic acid-responsive genes or light-induced stomatal opening signaling determinants, such as MYB60, MYB61, Hypersensitive to red and blue1, and Protein phosphatase7. CYCH;1 down-regulation induced the expression of redox homeostasis genes, such as LIPOXYGENASE3 (LOX3), LOX4, ARABIDOPSIS GLUTATHIONE PEROXIDASE 7 (ATGPX7), EARLY LIGHT-INDUCIBLE PROTEIN1 (ELIP1), and ELIP2, and increased hydrogen peroxide production in guard cells. Furthermore, loss-of-function mutations in CDKD;2 or CDKD;3 did not affect responsiveness to drought stress, suggesting that CYCH;1 regulates the drought stress response in a CDKD-independent manner. We propose that CYCH;1 regulates blue light-mediated stomatal opening by controlling reactive oxygen species homeostasis.
Assuntos
Arabidopsis/fisiologia , Ciclina H/metabolismo , Estômatos de Plantas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclina H/genética , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Secas , Regulação da Expressão Gênica de Plantas , Luz , Mutação , Transpiração Vegetal , Plantas Geneticamente Modificadas , Interferência de RNARESUMO
Eukaryotic cell division is controlled by cyclin-dependent kinases (CDKs), which require phosphorylation by a CDK-activating kinase (CAK) for full activity. Chemical genetics uncovered requirements for the metazoan CAK Cdk7 in determining cyclin specificity and activation order of Cdk2 and Cdk1 during S and G2 phases. It was unknown if Cdk7 also activates Cdk4 and Cdk6 to promote passage of the restriction (R) point, when continued cell-cycle progression becomes mitogen independent, or if CDK-activating phosphorylation regulates G1 progression. Here we show that Cdk7 is a Cdk4- and Cdk6-activating kinase in human cells, required to maintain activity, not just to establish the active state, as is the case for Cdk1 and Cdk2. Activating phosphorylation of Cdk7 rises concurrently with that of Cdk4 as cells exit quiescence and accelerates Cdk4 activation in vitro. Therefore, mitogen signaling drives a CDK-activation cascade during G1 progression, and CAK might be rate-limiting for R point passage.
Assuntos
Quinase 4 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Fase G1 , Processamento de Proteína Pós-Traducional , Motivos de Aminoácidos , Proliferação de Células , Ciclina D/metabolismo , Ciclina H/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/genética , Ativação Enzimática , Epistasia Genética , Células HCT116 , Humanos , Fosforilação , Proteína do Retinoblastoma/metabolismo , Fase S , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
BACKGROUND: Increased expression of cyclinH (CCNH) and cyclin-dependent kinase 7 (CDK7) has a relationship with poor prognosis in most human cancers. AIM: Investigate the expression of CCNH and CDK7 in human esophageal squamous cell carcinoma (ESCC) and the effect of chemotherapy on their expression. METHODS: Western blotting and immunohistochemistry were used to measure the expression of CCNH and CDK7 proteins in ESCC and adjacent normal tissue in 98 patients. We use Cell Counting Kit-8 and cell flow to analyze the effects of cisplatin and interference of CCNH and CDK7 in cell cycle process. RESULTS: Immunohistochemical analysis showed that CCNH and CDK7 expression were significantly associated with unfavorable clinicopathologic variables. CCNH and CDK7 protein levels were elevated in ESCC tissues in comparison with adjacent normal tissues. Survival analysis revealed that CCNH and CDK7 overexpression were significantly associated with overall survival (P < 0.001). Cisplatin or interference of CCNH or CDK7 led cells to grow slowly. Overexpression of CCNH and CDK7 in TE1 cells can lead to resistance to cisplatin. CONCLUSIONS: We can conclude that CCNH and CDK7 may play an important role in the tumorigenesis and development of ESCC. CCNH and CDK7 expression affected the chemotherapy of tumor.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Ciclina H/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Neoplasias Esofágicas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Western Blotting , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/mortalidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/mortalidade , Carcinoma de Células Escamosas do Esôfago , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Análise de Sobrevida , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Cells derived from the amniotic foetal membrane of human term placenta have drawn particular attention mainly for their plasticity and immunological properties, which render them interesting for stem-cell research and cell-based therapeutic applications. In particular, we have previously demonstrated that amniotic mesenchymal tissue cells (AMTC) inhibit lymphocyte proliferation in vitro and suppress the generation and maturation of monocyte-derived dendritic cells. Here, we show that AMTC also significantly reduce the proliferation of cancer cell lines of haematopoietic and non-haematopoietic origin, in both cell-cell contact and transwell co-cultures, therefore suggesting the involvement of yet-unknown inhibitory soluble factor(s) in this 'cell growth restraint'. Importantly, we provide evidence that the anti-proliferative effect of AMTC is associated with induction of cell cycle arrest in G0/G1 phase. Gene expression analyses demonstrate that AMTC can down-regulate cancer cells' mRNA expression of genes associated with cell cycle progression, such as cyclins (cyclin D2, cyclin E1, cyclin H) and cyclin-dependent kinase (CDK4, CDK6 and CDK2), whilst they up-regulate cell cycle negative regulator such as p15 and p21, consistent with a block in G0/G1 phase with no progression to S phase. Taken together, these findings warrant further studies to investigate the applicability of these cells for controlling cancer cell proliferation in vivo.
Assuntos
Âmnio/citologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Âmnio/metabolismo , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Técnicas de Cocultura , Ciclina D2/genética , Ciclina D2/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Ciclina H/genética , Ciclina H/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo , Fase G1/efeitos dos fármacos , Células HeLa , Humanos , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células U937 , Regulação para CimaRESUMO
CDK-activating kinase (CAK) phosphorylates threonine 161 (T161) of CDC2, a catalytic subunit of maturation/M-phase promoting factor (MPF), and is essential for MPF activation in mitosis. CAK has been thought to consist of a catalytic subunit, a regulatory subunit and an assembly factor: CDK7, CCNH (also known as cyclin H), and MNAT1 (also known as MAT1), respectively. Although it is known that the meiotic progression of oocytes is regulated by MPF activity, the role of CAK in meiosis is still unclear. In the present study, we attempted to confirm the involvement of CAK in the meiotic progression of porcine immature oocytes. The T161 phosphorylation of CDC2 was found around germinal vesicle breakdown (GVBD) and thereafter from 18 to 48 h of culture. The GVBD rate at 18 h was increased by the overexpression of CDC2 but not mutated CDC2 (T161 replaced by alanine). Transcripts of CDK7, CCNH, and MNAT1 were detectable throughout the culture period, and their protein distribution patterns during oocyte maturation were the same as those reported in mitotic somatic cells. Overexpression of CDK7 or CCNH accelerated the meiotic events, such as meiotic resumption, T161 phosphorylation of CDC2, CCNB (also known as Cyclin B) synthesis, and MPF activation. On the contrary, knockdown of CDK7 or CCNH caused the inhibition of these meiotic events. In contrast, overexpression and antisense RNA injection of MNAT1 had no influence on meiotic resumption, the status of T161 phosphorylation of CDC2, or MPF activity. These results suggest that CDK7 and CCNH activate CDC2 by T161 phosphorylation and make up CAK, which is required for normal meiotic progression during porcine oocyte maturation.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclina H/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Meiose , Oócitos/enzimologia , Animais , Proteínas de Transporte/metabolismo , Clonagem Molecular , Feminino , Expressão Gênica , Microinjeções , Fosforilação , RNA Mensageiro , Suínos , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Cyclin H regulates cell cycle transitions; it always forms trimeric cyclin-dependent protein kinases (CDK)-activating kinase (CAK) complex with CDK7 and MAT1 that phosphorylates a threonine residue in the CDK2 T loop region. However, neither the expression nor function of cyclin H in the central nervous system (CNS) injury is still clear. Therefore, we studied cyclin H in a rat spinal cord contusion model. Injury markedly increased cyclin H protein expression throughout the thoracic spinal cord but did not increase CDK7. However, double immunofluorescent staining for proliferating cell nuclear antigen (PCNA) and cell markers revealed increases of cyclin H and CDK2 in proliferating microglia and astrocytes, and the co-immunoprecipitation studies shown that the associations of cyclin H with CDK2 were enhanced evidently after injury. Our data suggest that cyclin H may play a proliferative role in spinal cord injury (SCI).
Assuntos
Ciclina H/metabolismo , Traumatismos da Medula Espinal/metabolismo , Medula Espinal/metabolismo , Animais , Biomarcadores/metabolismo , Ciclina H/genética , Quinase 2 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Masculino , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologia , Medula Espinal/patologia , Traumatismos da Medula Espinal/patologia , Fatores de Tempo , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Mouse B2 RNA represses RNA polymerase II (Pol II) transcription during the cellular heat shock response. B2 RNA binds Pol II, enters complexes at promoters, and keeps the polymerase from engaging the DNA. Here we show that phosphorylation of Ser5 residues in the Pol II carboxy terminal domain (CTD) decreases after heat shock at the promoter of the repressed actin gene in mouse cells, despite the continued presence of Cdk7 and cyclin H. Biochemical assays revealed that B2 RNA, when present with Pol II in promoter-bound complexes, specifically represses CTD phosphorylation by TFIIH.