RESUMO
The cancer syndrome polymerase proofreading-associated polyposis results from germline mutations in the POLE and POLD1 genes. Mutations in the exonuclease domain of these genes are associated with hyper- and ultra-mutated tumors with a predominance of base substitutions resulting from faulty proofreading during DNA replication. When a new variant is identified by gene testing of POLE and POLD1, it is important to verify whether the variant is associated with PPAP or not, to guide genetic counseling of mutation carriers. In 2015, we reported the likely pathogenic (class 4) germline POLE c.1373A > T p.(Tyr458Phe) variant and we have now characterized this variant to verify that it is a class 5 pathogenic variant. For this purpose, we investigated (1) mutator phenotype in tumors from two carriers, (2) mutation frequency in cell-based mutagenesis assays, and (3) structural consequences based on protein modeling. Whole-exome sequencing of two tumors identified an ultra-mutator phenotype with a predominance of base substitutions, the majority of which are C > T. A SupF mutagenesis assay revealed increased mutation frequency in cells overexpressing the variant of interest as well as in isogenic cells encoding the variant. Moreover, exonuclease repair yeast-based assay supported defect in proofreading activity. Lastly, we present a homology model of human POLE to demonstrate structural consequences leading to pathogenic impact of the p.(Tyr458Phe) mutation. The three lines of evidence, taken together with updated co-segregation and previously published data, allow the germline variant POLE c.1373A > T p.(Tyr458Phe) to be reclassified as a class 5 variant. That means the variant is associated with PPAP.
Assuntos
DNA Polimerase II , Neoplasias , Humanos , DNA Polimerase II/genética , DNA Polimerase II/química , DNA Polimerase II/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Neoplasias/genética , Mutação , Exonucleases/genética , Exonucleases/metabolismoRESUMO
PURPOSE: Germline pathogenic variants in the exonuclease domain (ED) of polymerases POLE and POLD1 predispose to adenomatous polyps, colorectal cancer (CRC), endometrial tumors, and other malignancies, and exhibit increased mutation rate and highly specific associated mutational signatures. The tumor spectrum and prevalence of POLE and POLD1 variants in hereditary cancer are evaluated in this study. METHODS: POLE and POLD1 were sequenced in 2813 unrelated probands referred for genetic counseling (2309 hereditary cancer patients subjected to a multigene panel, and 504 patients selected based on phenotypic characteristics). Cosegregation and case-control studies, yeast-based functional assays, and tumor mutational analyses were performed for variant interpretation. RESULTS: Twelve ED missense variants, 6 loss-of-function, and 23 outside-ED predicted-deleterious missense variants, all with population allele frequencies <1%, were identified. One ED variant (POLE p.Met294Arg) was classified as likely pathogenic, four as likely benign, and seven as variants of unknown significance. The most commonly associated tumor types were colorectal, endometrial and ovarian cancers. Loss-of-function and outside-ED variants are likely not pathogenic for this syndrome. CONCLUSIONS: Polymerase proofreading-associated syndrome constitutes 0.1-0.4% of familial cancer cases, reaching 0.3-0.7% when only CRC and polyposis are considered. ED variant interpretation is challenging and should include multiple pieces of evidence.
Assuntos
Neoplasias Colorretais , DNA Polimerase II , DNA Polimerase II/genética , DNA Polimerase III , Mutação em Linhagem Germinativa , Humanos , Mutação , Proteínas de Ligação a Poli-ADP-Ribose/genéticaRESUMO
We describe a family in which four siblings exhibited multiple or classic colonic polyposis with or without colorectal carcinoma (CRC). One female developed three primary tumors, including CRC and carcinomas of the ovary and breast. Whole-exome sequencing of germline DNA from affected and unaffected individuals revealed a novel missense mutation in the exonuclease domain of POLE (c.833C>A; p.Thr278Lys) associated with a highly penetrant, autosomal-dominant inheritance pattern. Functional studies in yeast and demonstration of a high mutational burden in the available tumors confirmed the pathogenicity of the novel variant. Prominent POLE-deficient somatic mutational signatures were seen in the CRCs, but in contrast, a mutational signature typical of concomitant tumoral loss of POLE and mismatch-repair function (POLE-exo* /MSI) was noted in the breast cancer. The breast cancer also showed distinctive pathological characteristics that reflect the presence of both the germline POLE variant and the secondary somatic MMR alterations.
Assuntos
DNA Polimerase II/genética , Mutação em Linhagem Germinativa/genética , Mutação/genética , Neoplasias Primárias Múltiplas/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Sequência de Bases , Família , Feminino , Humanos , Masculino , Neoplasias Primárias Múltiplas/patologia , LinhagemRESUMO
The cyclin-dependent kinase inhibitor p27Kip1 (p27) also behaves as a transcriptional repressor. Data showing that the p300/CBP-associated factor (PCAF) acetylates p27 inducing its degradation suggested that PCAF and p27 could collaborate in the regulation of transcription. However, this possibility remained to be explored. We analyzed here the transcriptional programs regulated by PCAF and p27 in the colon cancer cell line HCT116 by chromatin immunoprecipitation sequencing (ChIP-seq). We identified 269 protein-encoding genes that contain both p27 and PCAF binding sites being the majority of these sites different for PCAF and p27. PCAF or p27 knock down revealed that both regulate the expression of these genes, PCAF as an activator and p27 as a repressor. The double knock down of PCAF and p27 strongly reduced their expression indicating that the activating role of PCAF overrides the repressive effect of p27. We also observed that the transcription factor Pax5 interacts with both p27 and PCAF and that the knock down of Pax5 induces the expression of p27/PCAF target genes indicating that it also participates in the transcriptional regulation mediated by p27/PCAF. In summary, we report here a previously unknown mechanism of transcriptional regulation mediated by p27, Pax5 and PCAF.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/fisiologia , Regulação da Expressão Gênica , Fator de Transcrição PAX5/fisiologia , Fatores de Transcrição de p300-CBP/fisiologia , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Células Cultivadas , Cromatina/metabolismo , Células HCT116 , Humanos , Células MCF-7 , Camundongos , Ligação Proteica , Proteínas/genética , Análise Serial de Tecidos , Transcrição GênicaRESUMO
Transcriptional repressor complexes containing p130 and E2F4 regulate the expression of genes involved in DNA replication. During the G1 phase of the cell cycle, sequential phosphorylation of p130 by cyclin-dependent kinases (Cdks) disrupts these complexes allowing gene expression. The Cdk inhibitor and tumor suppressor p27(Kip1) associates with p130 and E2F4 by its carboxyl domain on the promoters of target genes but its role in the regulation of transcription remains unclear. We report here that p27(Kip1) recruits cyclin D2/D3-Cdk4 complexes on the promoters by its amino terminal domain in early and mid G1. In cells lacking p27(Kip1), cyclin D2/D3-Cdk4 did not associate to the promoters and phosphorylation of p130 and transcription of target genes was increased. In late G1, these complexes were substituted by p21(Cip1)-cyclin D1-Cdk2. In p21(Cip1) null cells cyclin D1-Cdk2 were not found on the promoters and transcription was elevated. In p21/p27 double null cells transcription was higher than in control cells and single knock out cells. Thus, our results clarify the role of p27(Kip1) and p21(Cip1) in transcriptional regulation of genes repressed by p130/E2F4 complexes in which p27(Kip1) and p21(Cip1) play a sequential role by recruiting and regulating the activity of specific cyclin-Cdk complexes on the promoters.
Assuntos
Ciclina D/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Aurora Quinase A/metabolismo , Células Cultivadas , Ciclina D2/metabolismo , Ciclina D3/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Fase G1/genética , Camundongos , Mutação , Células NIH 3T3 , Proteínas Repressoras/metabolismoRESUMO
Calcium signals trigger the translocation of the Prz1 transcription factor from the cytoplasm to the nucleus. The process is regulated by the calcium-activated phosphatase calcineurin, which activates Prz1 thereby maintaining active transcription during calcium signalling. When calcium signalling ceases, Prz1 is inactivated by phosphorylation and exported to the cytoplasm. In budding yeast and mammalian cells, different kinases have been reported to counter calcineurin activity and regulate nuclear export. Here, we show that the Ca(2+)/calmodulin-dependent kinase Cmk1 is first phosphorylated and activated by the newly identified kinase CaMKK2 homologue, Ckk2, in response to Ca(2+). Then, active Cmk1 binds, phosphorylates and inactivates Prz1 transcription activity whilst at the same time cmk1 expression is enhanced by Prz1 in response to Ca(2+). Furthermore, Cdc25 phosphatase is also phosphorylated by Cmk1, inducing cell cycle arrest in response to an increase in Ca(2+). Moreover, cmk1 deletion shows a high tolerance to chronic exposure to Ca(2+), due to the lack of cell cycle inhibition and elevated Prz1 activity. This work reveals that Cmk1 kinase activated by the newly identified Ckk2 counteracts calcineurin function by negatively regulating Prz1 activity which in turn is involved in activating cmk1 gene transcription. These results are the first insights into Cmk1 and Ckk2 function in Schizosaccharomyces pombe.
Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Transcrição/metabolismo , Cálcio , Sinalização do Cálcio , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/biossíntese , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/biossíntese , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Pontos de Checagem do Ciclo Celular , Retroalimentação Fisiológica , Deleção de Genes , Pressão Osmótica , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/biossíntese , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/genéticaRESUMO
The fission yeast mitogen-activated kinase (MAPK) Sty1 is essential for cell survival in response to different environmental insults. In unstimulated cells, Sty1 forms an inactive ternary cytoplasmatic complex with the MAPKK Wis1 and the MAPKAP kinase Srk1. Wis1 phosphorylates and activates Sty1, inducing the nuclear translocation of the complex. Once in the nucleus, Sty1 phosphorylates and activates Srk1, which in turns inhibits Cdc25 and cell cycle progression, before being degraded in a proteasome-dependent manner. In parallel, active nuclear Sty1 activates the transcription factor Atf1, which results in the expression of stress response genes including pyp2 (a MAPK phosphatase) and srk1. Despite its essentiality in response to stress, persistent activation of the MAPK pathway can be deleterious and induces cell death. Thus, timely pathway inactivation is essential to ensure an appropriate response and cell viability. Here, uncover a role for the MAPKAP kinase Srk1 as an essential component of a negative feedback loop regulating the Sty1 pathway through phosphorylation and inhibition of the Wis1 MAPKK. This feedback regulation by a downstream kinase in the pathway highlights an additional mechanism for fine-tuning of MAPK signaling. Thus, our results indicate that Srk1 not only facilitates the adaptation to stress conditions by preventing cell cycle progression, but also plays an instrumental role regulating the upstream kinases in the stress MAPK pathway.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Retroalimentação , Regulação Fúngica da Expressão Gênica , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mitógenos/metabolismo , Fosforilação , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
This work aimed to evaluate the phytochemical content and to determine the antioxidant and cytotoxic activities of methanol extracts of the carob tree (Ceratonia siliqua L.) germ flour. The extracts were rich in phenolic compounds, had considerable antioxidant activity, and reduced the viability of cervical (HeLa) cancer cells. The chemical content and the biological activities of the extracts were significantly affected by gender and cultivar. Female cultivar Galhosa had the highest levels of phenolic compounds, and the highest antioxidant activity. Extracts from the hermaphrodite trees and from the female cultivars Galhosa and Costela/Canela exhibited the highest cytotoxic activity. The most abundant compound was theophylline. The phenolic content was correlated to both antioxidant and cytotoxic activities. Our findings provide new knowledge about the health implications of consuming food supplemented with carob germ flour.
Assuntos
Antioxidantes/farmacologia , Fabaceae/química , Galactanos/farmacologia , Mananas/farmacologia , Extratos Vegetais/farmacologia , Gomas Vegetais/farmacologia , Teofilina/farmacologia , Galactanos/química , Células HeLa , Humanos , Mananas/química , Fenóis/análise , Fenóis/farmacologia , Extratos Vegetais/análise , Gomas Vegetais/química , Teofilina/análiseRESUMO
RNA-binding proteins (RBPs) play a major role during control of mRNA localization, stability, and translation and are central to most cellular processes. In the fission yeast Schizosaccharomyces pombe, the multiple K homology (KH) domain RBP Rnc1 downregulates the activity of the cell integrity pathway (CIP) via stabilization of pmp1+ mRNA, which encodes the Pmp1 phosphatase that inactivates Pmk1, the mitogen-activated protein kinase (MAPK) component of this signaling cascade. However, Rnc1 likely regulates the half-life/stability of additional mRNAs. We show that Rnc1 downregulates the activity of Sty1, the MAPK of the stress-activated MAPK pathway (SAPK), during control of cell length at division and recovery in response to acute stress. Importantly, this control strictly depends on Rnc1's ability to bind mRNAs encoding activators (Wak1 MAPKKK, Wis1 MAPKK) and downregulators (Atf1 transcription factor, Pyp1 and Pyp2 phosphatases) of Sty1 phosphorylation through its KH domains. Moreover, Sty1 is responsible for Rnc1 phosphorylation in vivo at multiple phosphosites during growth and stress, and these modifications trigger Rnc1 for proper binding and destabilization of the above mRNA targets. Phosphorylation by Sty1 prompts Rnc1-dependent mRNA destabilization to negatively control SAPK signaling, thus revealing an additional feedback mechanism that allows precise tuning of MAPK activity during unperturbed cell growth and stress.IMPORTANCE Control of mRNA localization, stability, turnover, and translation by RNA-binding proteins (RBPs) influences essential processes in all eukaryotes, including signaling by mitogen-activated protein kinase (MAPK) pathways. We describe that in the fission yeast Schizosaccharomyces pombe the RBP Rnc1 negatively regulates cell length at division during unperturbed growth and recovery after acute stress by reducing the activity of the MAPK Sty1, which regulates cell growth and differentiation during environmental cues. This mechanism relies on Rnc1 binding to specific mRNAs encoding both enhancers and negative regulators of Sty1 activity. Remarkably, multiple phosphorylation of Rnc1 by Sty1 favors RBP binding and destabilization of the above mRNAs. Thus, posttranscriptional modulation of MAP kinase signaling by RNA-binding proteins emerges as a major regulatory mechanism that dictates the growth cycle and cellular adaptation in response to the changing environment in eukaryotic organisms.
Assuntos
Desoxirribonucleases/metabolismo , Retroalimentação Fisiológica , Sistema de Sinalização das MAP Quinases , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiologia , Estresse Fisiológico , Desoxirribonucleases/genética , Modelos Biológicos , Mutação , Fosforilação , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Mitogenic response to growth factors is concomitant with the modulation they exert on the levels of Fructose 2,6-bisphosphate (Fru-2,6-P2), an essential activator of the glycolytic flux. In mammalian cells, decreased Fru-2,6-P2 concentration causes cell cycle delay, whereas high levels of Fru-2,6-P2 sensitize cells to apoptosis. In order to analyze the cell cycle consequences due to changes in Fru-2,6-P2 levels, the bisphosphatase-dead mutant (H258A) of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase enzyme was over-expressed in Schizosaccharomyces pombe cells and the variation in cell phenotype was studied. The results obtained demonstrate that the increase in Fru-2,6-P2 levels results in a defective division of S. pombe, as revealed by an altered multisepted phenotype. The H258A-expressing cells showed impairment of cytokinesis, but normal nuclear division. In order to identify cellular mediators responsible for this effect, we transformed different S. pombe strains and observed that the cytokinetic defect was absent in cells defective for Wee1 kinase function. Therefore, in S. pombe, Wee1 integrates the metabolic signal emerging from changes in Fru-2,6-P2 content, thus coupling metabolism with cell proliferation. As the key regulators of the cell cycle checkpoints are conserved throughout evolution, these results may help to understand the experimental evidences obtained by manipulation of Fru-2,6-P2 levels in mammalian cells.
Assuntos
Frutosedifosfatos/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Divisão Celular , Regulação Fúngica da Expressão Gênica , Mutação/genética , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
The nucleosome assembly protein Nap1 has been implicated in various cellular functions such as histone shuttling into the nucleus, nucleosome assembly, chromatin remodelling, transcriptional control and cell-cycle regulation in Saccharomyces cerevisiae. In Schizosaccharomyces pombe nap1 null mutant cells are viable but they showed a delay in the onset of mitosis which is rescued by the absence of the replication Cds1 checkpoint kinase. In contrast, the absence of the DNA-damage Chk1 checkpoint kinase is unable to rescue the delay. Moreover, the double nap1 cds1 mutant cells lose viability and cells show positive H2AX phosphorylation, suggesting that the viability of nap1-deleted cells is due to the Cds1 kinase. We also show that overexpression of Nap1 protein blocks the cell cycle in G1 phase.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Proliferação de Células , Sobrevivência Celular , Quinase do Ponto de Checagem 2 , Cromatina/metabolismo , Citoesqueleto/metabolismo , Replicação do DNA , Fase G1 , Deleção de Genes , Instabilidade Genômica , Mitose , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Homologia de Sequência de AminoácidosRESUMO
Herein is reported the design and synthesis of poly(ethylene glycol) derivatives of Lamellarin D with the aim of modulating their physicochemical properties and improving the biological activity. Mono-, di-, and tri-PEG conjugates with improved solubility were obtained in 18-57% overall yields from the corresponding partially protected phenolic derivatives of Lamellarin D. Conjugates 1-9 were tested in a panel of three human tumor cell lines (MDA-MB-231 breast, A-549 lung, and HT-29 colon) to evaluate their cytotoxicity. Several compounds exhibited enhanced cellular internalization, and more than 85% of the derivatives showed a lower GI(50) than Lam-D. Furthermore, cell cycle arrest at G2 phase and apoptotic cell-death pathways were determined for Lamellarin D and these derivatives.
Assuntos
Antineoplásicos/química , Antineoplásicos/metabolismo , Cumarínicos/química , Cumarínicos/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Isoquinolinas/química , Isoquinolinas/metabolismo , Polietilenoglicóis/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Transporte Biológico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cumarínicos/síntese química , Cumarínicos/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/síntese química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Isoquinolinas/síntese química , Isoquinolinas/farmacologiaRESUMO
The design and synthesis of Lamellarin D conjugates with a nuclear localization signal peptide and a poly(ethylene glycol)-based dendrimer are described. Conjugates 1-4 were obtained in 8-84% overall yields from the corresponding protected Lamellarin D. Conjugates 1 and 4 are 1.4- to 3.3-fold more cytotoxic than the parent compound against three human tumor cell lines (MDA-MB-231 breast, A-549 lung, and HT-29 colon). Besides, conjugates 3 and 4 showed a decrease in activity potency in BJ skin fibroblasts, a normal cell culture. Cellular internalization was analyzed, and a nuclear distribution pattern was observed for 4, which contains a nuclear localization signaling sequence.
Assuntos
Antineoplásicos/química , Antineoplásicos/metabolismo , Cumarínicos/química , Cumarínicos/metabolismo , Dendrímeros/química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Isoquinolinas/química , Isoquinolinas/metabolismo , Sinais de Localização Nuclear/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Transporte Biológico , Linhagem Celular Tumoral , Cumarínicos/síntese química , Cumarínicos/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/síntese química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Isoquinolinas/síntese química , Isoquinolinas/farmacologia , Polietilenoglicóis/química , TransfecçãoRESUMO
GSK3beta and E2F1 play an important role in the control of proliferation and apoptosis. Previous work has demonstrated that GSK3beta indirectly regulates E2F activity through modulation of cyclin D1 levels. In this work we show that GSK3beta phosphorylates human E2F1 in vitro at serine 403 and threonine 433, both residues localized at its transactivation domain. This phosphorylation was not detected in vivo. However, co-immunoprecipitation experiments do reveal in vivo binding of these proteins. Moreover, uninhibitable and catalitycally inactive GSK3beta forms inhibit the transcriptional activity of a fusion protein containing E2F1 transactivation domain. Both forms of GSK3beta inhibit E2F1 with similar efficiency. Interestingly the effect was independent of the mutation of serine 403 and threonine 433 to alanine. This suggests that this transcriptional modulation is independent of GSK3beta kinase activity and phosphorylation state of serine 403 and threonine 433. The re-targeting of these GSK3beta forms to the nucleus results in a higher capacity to regulate E2F1 transcriptional activity. Depletion of the levels of GSK3beta protein using siRNA activates E2F1 transcriptional activity. The data presented in this study offer a new mechanism of regulation of E2F1 by direct binding of GSK3beta to its transactivation domain.
Assuntos
Fator de Transcrição E2F1/metabolismo , Regulação Enzimológica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Transativadores/metabolismo , Linhagem Celular , Fator de Transcrição E2F1/genética , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , Transativadores/genéticaRESUMO
Different studies have demonstrated multiple effects of arsenite on human physiology. However, there are many open questions concerning the mechanism of response to arsenite. Schizosaccharomyces pombe activates the Sty1 MAPK pathway as a common response to several stress conditions. The specificity of the response is due to the activation of different transcription factors and specific targets such the Cmk2 MAPKAP kinase. We have previously shown that Cmk2 is phosphorylated and activated by the MAPK Sty1 in response to oxidative stress. Here, we report that Cmk2 kinase is specifically necessary to overcome the stress caused by metalloid agents, in particular arsenite. Deletion of cmk2 increases the protein level of various components of the MAPK pathway. Moreover, Cmk2 negatively regulates translation through the Cpc2 kinase: the RACK1 orthologue in fission yeast. RACK1 is a receptor for activated C-kinase. Interestingly, RACK1 is a constituent of the eukaryotic ribosome specifically localized in the head region of the 40â¯S subunit. Cmk2 controls arsenite response through Cpc2 and it does so through Cpc2 ribosomal function, as observed in genetic analysis using a Cpc2 mutant unable to bind to ribosome. These findings suggest a role for Cmk2 in regulating translation and facilitating adaptation to arsenite stress in the ribosome.
Assuntos
Arsenitos/toxicidade , Regulação Fúngica da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , Receptores de Quinase C Ativada/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/efeitos dos fármacos , DNA Fúngico/genética , DNA Fúngico/metabolismo , Viabilidade Microbiana/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/deficiência , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Receptores de Quinase C Ativada/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Transdução de Sinais , Estresse FisiológicoRESUMO
Alpha-synuclein (α-SYN) is the main component of anomalous protein aggregates (Lewy bodies) that play a crucial role in several neurodegenerative diseases (synucleinopathies) like Parkinson's disease and multiple system atrophy. However, the mechanisms involved in its transcriptional regulation are poorly understood. We investigated here the role of the cyclin-dependent kinase (Cdk) inhibitor and transcriptional regulator p27Kip1 (p27) in the regulation of α-SYN expression. We observed that selective deletion of p27 by CRISPR/Cas9 technology in neural cells resulted in increased levels of α-SYN. Knock-down of the member of the same family p21Cip1 (p21) also led to increased α-SYN levels, indicating that p27 and p21 collaborate in the repression of α-SYN transcription. We demonstrated that this repression is mediated by the transcription factor E2F4 and the member of the retinoblastoma protein family p130 and that it is dependent of Cdk activity. Chromatin immunoprecipitation analysis revealed specific binding sites for p27, p21 and E2F4 in the proximal α-SYN gene promoter. Finally, luciferase assays revealed a direct action of p27, p21 and E2F4 in α-SYN gene expression. Our findings reveal for the first time a negative regulatory mechanism of α-SYN expression, suggesting a putative role for cell cycle regulators in the etiology of synucleinopathies.
RESUMO
The protein p27Kip1 is a member of the Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors. It interacts with both the catalytic and the regulatory subunit (cyclin) and introduces a region into the catalytic cleave of the Cdk inducing its inactivation. Its inhibitory capacity can be modulated by specific tyrosine phosphorylations. p27Kip1 also behaves as a transcriptional regulator. It associates with specific chromatin domains through different transcription factors. ChIP on chip, ChIP-seq and expression microarray analysis allowed the identification of the transcriptional programs regulated by p27Kip1. Thus, important cellular functions as cell division cycle, respiration, RNA processing, translation and cell adhesion, are under p27Kip1 regulation. Moreover, genes involved in pathologies as cancer and neurodegeneration are also regulated by p27Kip1, suggesting its implication in these pathologies. The carboxyl moiety of p27Kip1 can associate with different proteins, including transcriptional regulators. In contrast, its NH2-terminal region specifically interacts with cyclin-Cdk complexes. The general mechanistic model of how p27Kip1 regulates transcription is that it associates by its COOH region to the transcriptional regulators on the chromatin and by the NH2-domain to cyclin-Cdk complexes. After Cdk activation it would phosphorylate the specific targets on the chromatin leading to gene expression. This model has been demonstrated to apply in the transcriptional regulation of p130/E2F4 repressed genes involved in cell cycle progression. We summarize in this review our current knowledge on the role of p27Kip1 in the regulation of transcription, on the transcriptional programs under its regulation and on its relevance in pathologies as cancer and neurodegeneration.
RESUMO
The protein p27Kip1 (p27), a member of the Cip-Kip family of cyclin-dependent kinase inhibitors, is involved in tumorigenesis and a correlation between reduced levels of this protein in human tumours and a worse prognosis has been established. Recent reports revealed that p27 also behaves as a transcriptional regulator. Thus, it has been postulated that the development of tumours with low amounts of p27 could be propitiated by deregulation of transcriptional programs under the control of p27. However, these programs still remain mostly unknown. The aim of this study has been to define the transcriptional programs regulated by p27 by first identifying the p27-binding sites (p27-BSs) on the whole chromatin of quiescent mouse embryonic fibroblasts. The chromatin regions associated to p27 have been annotated to the most proximal genes and it has been considered that the expression of these genes could by regulated by p27. The identification of the chromatin p27-BSs has been performed by Chromatin Immunoprecipitation Sequencing (ChIP-seq). Results revealed that p27 associated with 1839 sites that were annotated to 1417 different genes being 852 of them protein coding genes. Interestingly, most of the p27-BSs were in distal intergenic regions and introns whereas, in contrast, its association with promoter regions was very low. Gene ontology analysis of the protein coding genes revealed a number of relevant transcriptional programs regulated by p27 as cell adhesion, intracellular signalling and neuron differentiation among others. We validated the interaction of p27 with different chromatin regions by ChIP followed by qPCR and demonstrated that the expressions of several genes belonging to these programs are actually regulated by p27. Finally, cell adhesion assays revealed that the adhesion of p27-/- cells to the plates was much higher that controls, revealing a role of p27 in the regulation of a transcriptional program involved in cell adhesion.
Assuntos
Cromatina/química , Inibidor de Quinase Dependente de Ciclina p27/genética , DNA Intergênico/genética , Fibroblastos/metabolismo , Genoma , Transcrição Gênica , Animais , Sítios de Ligação , Adesão Celular , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , DNA Intergênico/metabolismo , Embrião de Mamíferos , Fibroblastos/citologia , Regulação da Expressão Gênica , Ontologia Genética , Células HCT116 , Humanos , Camundongos , Anotação de Sequência Molecular , Cultura Primária de Células , Regiões Promotoras Genéticas , Ligação Proteica , Análise de Sequência de DNA , Transdução de SinaisRESUMO
Germline mutations in POLE and POLD1 have been shown to cause predisposition to colorectal multiple polyposis and a wide range of neoplasms, early-onset colorectal cancer being the most prevalent. In order to find additional mutations affecting the proofreading activity of these polymerases, we sequenced its exonuclease domain in 155 patients with multiple polyps or an early-onset colorectal cancer phenotype without alterations in the known hereditary colorectal cancer genes. Interestingly, none of the previously reported mutations in POLE and POLD1 were found. On the other hand, among the genetic variants detected, only two of them stood out as putative pathogenic in the POLE gene, c.1359 + 46del71 and c.1420G > A (p.Val474Ile). The first variant, detected in two families, was not proven to alter correct RNA splicing. Contrarily, c.1420G > A (p.Val474Ile) was detected in one early-onset colorectal cancer patient and located right next to the exonuclease domain. The pathogenicity of this change was suggested by its rarity and bioinformatics predictions, and it was further indicated by functional assays in Schizosaccharomyces pombe. This is the first study to functionally analyze a POLE genetic variant outside the exonuclease domain and widens the spectrum of genetic changes in this DNA polymerase that could lead to colorectal cancer predisposition.
Assuntos
Polipose Adenomatosa do Colo/diagnóstico , Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , DNA Polimerase III/genética , DNA Polimerase II/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Adolescente , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Alelos , Substituição de Aminoácidos , Criança , Pré-Escolar , Neoplasias Colorretais/prevenção & controle , DNA Polimerase II/química , DNA Polimerase III/química , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Testes Genéticos , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Mutação , Linhagem , Proteínas de Ligação a Poli-ADP-Ribose/química , Conformação Proteica , Domínios Proteicos/genética , Adulto JovemRESUMO
BACKGROUND: Calcium/calmodulin-dependent protein kinase kinase (CaMKK) is required for diverse cellular functions. Mammalian CaMKK activates CaMKs and also the evolutionarily-conserved AMP-activated protein kinase (AMPK). The fission yeast Schizosaccharomyces pombe CaMKK, Ssp1, is required for tolerance to limited glucose through the AMPK, Ssp2, and for the integration of cell growth and division through the SAD kinase Cdr2. RESULTS: Here we report that Ssp1 controls the G2/M transition by regulating the activity of the CaMK Srk1. We show that inhibition of Cdc25 by Srk1 is regulated by Ssp1; and also that restoring growth polarity and actin localization of ssp1-deleted cells by removing the actin-monomer-binding protein, twinfilin, is sufficient to suppress the ssp1 phenotype. CONCLUSIONS: These findings demonstrate that entry into mitosis is mediated by a network of proteins, including the Ssp1 and Srk1 kinases. Ssp1 connects the network of components that ensures proper polarity and cell size with the network of proteins that regulates Cdk1-cyclin B activity, in which Srk1 plays an inhibitory role.