RESUMO
Estrogen receptor α (ERα) is the key transcriptional driver in a large proportion of breast cancers. We report that APOBEC3B (A3B) is required for regulation of gene expression by ER and acts by causing C-to-U deamination at ER binding regions. We show that these C-to-U changes lead to the generation of DNA strand breaks through activation of base excision repair (BER) and to repair by non-homologous end-joining (NHEJ) pathways. We provide evidence that transient cytidine deamination by A3B aids chromatin modification and remodelling at the regulatory regions of ER target genes that promotes their expression. A3B expression is associated with poor patient survival in ER+ breast cancer, reinforcing the physiological significance of A3B for ER action.
Assuntos
Neoplasias da Mama/genética , Citidina Desaminase/genética , Citidina/metabolismo , Reparo do DNA por Junção de Extremidades , Receptor alfa de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Sítios de Ligação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Citidina Desaminase/antagonistas & inibidores , Citidina Desaminase/metabolismo , DNA/genética , DNA/metabolismo , Dano ao DNA , Desaminação , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Antígenos de Histocompatibilidade Menor , Prognóstico , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida , Transcrição Gênica , Fator Trefoil-1 , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The E3 ubiqutin ligase, murne double-minute clone 2 (MDM2), promotes the degradation of p53 under normal homeostatic conditions. Several serine residues within the acidic domain of MDM2 are phosphorylated to maintain its activity but become hypo-phosphorylated following DNA damage, leading to inactivation of MDM2 and induction of p53. However, the signalling pathways that mediate these phosphorylation events are not fully understood. Here we show that the oncogenic and cell cycle-regulatory protein kinase, polo-like kinase-1 (PLK1), phosphorylates MDM2 at one of these residues, Ser260, and stimulates MDM2-mediated turnover of p53. These data are consistent with the idea that deregulation of PLK1 during tumourigenesis may help suppress p53 function.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Serina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação/genética , Western Blotting , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Células HeLa , Humanos , Imunoprecipitação , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Transfecção , Proteína Supressora de Tumor p53/genética , Quinase 1 Polo-LikeRESUMO
The p53 tumour suppressor protein is tightly controlled by the E3 ubiquitin ligase, mouse double minute 2 (MDM2), but maintains MDM2 expression as part of a negative feedback loop. We have identified the immunophilin, 25kDa FK506-binding protein (FKBP25), previously shown to be regulated by p53-mediated repression, as an MDM2-interacting partner. We show that FKBP25 stimulates auto-ubiquitylation and proteasomal degradation of MDM2, leading to the induction of p53. Depletion of FKBP25 by siRNA leads to increased levels of MDM2 and a corresponding reduction in p53 and p21 levels. These data are consistent with the idea that FKBP25 contributes to regulation of the p53-MDM2 negative feedback loop.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Escherichia coli/genética , Genes Reporter , Glutationa Transferase/metabolismo , Células HCT116 , Humanos , Luciferases/metabolismo , Camundongos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Ligação a Tacrolimo/genética , Transfecção , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Phospholipase C (PLC) and the small G protein RhoA are vital elements for the contraction of vascular smooth muscle cells. The available evidence points to altered PLC-delta1 activity as an element determining enhanced vascular tone in hypertension; however, the factor(s) responsible for increased PLC activity remains unknown. There is the data indicating that RhoA inhibits PLC-delta1 and factors downmodulating RhoA activate phospholipase. In the present study, we explore an impact of a newly identified human ARHGAP6 protein possessing GTPase stimulating activity for RhoA on the catalytic properties of PLC-delta1. Under in vitro conditions, ARHGAP6 protein activated PLC-delta1. ARHGAP6 protein bound PLC-delta1 and regulated its activity by masking the binding sites for inhibitory phospholipids. Moreover, ARHGAP6 increased the V(max) of PLC-delta1 and enhanced its response to Ca(2+) stimulation. A Western blot of immunoprecipitates from Cos-7 cells transfected with pcDNA3-ARHGAP6 and pcDNA3-PLCdelta1 showed the presence of ARHGAP6/PLC-delta1 complexes. The activity of PLC in cells overexpressing ARHGAP6 increased approximately 6-fold compared to control cells. The examination of ARHGAP6 expression in mononuclear cells isolated from the blood of patients with hypertension showed increased ARHGAP6 mRNA and protein levels compared to age-matched normotensive subjects. Enhanced expression of ARHGAP6 was associated with an elevated level of PLC activity and increased levels of IP(3) (1.6-fold) and DAG (2.3-fold). In summary, our data indicate that ARHGAP6 protein binds to and up regulates PLC-delta1 both under in vitro and in vivo conditions. Moreover, the elevated expression of ARHGAP6 provides possible explanation for the altered activity of PLC-delta1 in hypertension.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Hipertensão/enzimologia , Fosfolipase C delta/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Adulto , Animais , Western Blotting , Células COS , Chlorocebus aethiops , Diglicerídeos/metabolismo , Feminino , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Cinética , Leucócitos Mononucleares/metabolismo , Masculino , Fosfolipídeos/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
In this report we describe cloning and expression of human Rho GTPase activating protein (ARHGAP6) isoform 4 in Escherichia coli cells as a fusion protein with 6xHis. We cloned the ARHGAP6 cDNA into the bacterial expression vector pPROEX-1. Induction of the 6xHis-ARHGAP6 protein in BL21(DE3) and DH5alpha cells caused lysis of the cells irrespective of the kind of culture medium used. Successful expression of the fusion protein was obtained in the MC4100Deltaibp mutant strain lacking the small heat-shock proteins IbpA and IbpB. Reasonable yield was obtained when the cells were cultured in Terrific Broth + 1% glucose medium at 22 degrees C for 16 h. The optimal cell density for expression of soluble 6xHis-ARHGAP6 protein was at A(600) about 0.5. Under these conditions over 90% of the fusion protein was present in a soluble form. The 6xHis-ARHGAP6 protein was purified to near homogeneity by a two step procedure comprising chromatography on Ni-nitrilotriacetate and cation exchange columns. The expression system and purification procedure employed made it possible to obtain 1-2 mg of pure 6xHis-ARHGAP6 protein from 300 ml (1.5 g of cells) of E. coli culture.
Assuntos
Proteínas Ativadoras de GTPase/genética , Especificidade de Anticorpos , Sequência de Bases , Clonagem Molecular , Primers do DNA , Escherichia coli/genética , Proteínas Ativadoras de GTPase/isolamento & purificação , Proteínas Ativadoras de GTPase/metabolismo , Vetores Genéticos , Histidina , Humanos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/isolamento & purificação , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Phospholipase C (PLC, EC 3.1.4.11) is an enzyme crucial for the phosphoinositol pathway and whose activity is involved in eukaryotic signal transduction as it generates two second messengers: diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). There are four major types of phospholipase C named: beta, gamma, delta and the recently discovered epsilon, but this review will focus only on the recent advances for the delta isozymes of PLC. So far, four delta isozymes (named delta1-4) have been discovered and examined. They differ with regard to cellular distribution, activities, biochemical features and involvement in human ailments.
