RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most intractable and devastating malignant tumors. Epigenetic modifications such as DNA methylation and histone modification regulate tumor initiation and progression. However, the contribution of histone variants in PDAC is unknown. Here, we demonstrated that the histone variant H2A.Z is highly expressed in PDAC cell lines and PDAC patients and that its overexpression correlates with poor prognosis. Moreover, all three H2A.Z isoforms (H2A.Z.1, H2A.Z.2.1, and H2A.Z.2.2) are highly expressed in PDAC cell lines and PDAC patients. Knockdown of these H2A.Z isoforms in PDAC cell lines induces a senescent phenotype, cell cycle arrest in phase G2/M, increased expression of cyclin-dependent kinase inhibitor CDKN2A/p16, SA-ß-galactosidase activity and interleukin 8 production. Transcriptome analysis of H2A.Z-depleted PDAC cells showed altered gene expression in fatty acid biosynthesis pathways and those that regulate cell cycle and DNA damage repair. Importantly, depletion of H2A.Z isoforms reduces the tumor size in a mouse xenograft model in vivo and sensitizes PDAC cells to gemcitabine. Overexpression of H2A.Z.1 and H2A.Z.2.1 more than H2A.Z.2.2 partially restores the oncogenic phenotype. Therefore, our data suggest that overexpression of H2A.Z isoforms enables cells to overcome the oncoprotective barrier associated with senescence, favoring PDAC tumor grow and chemoresistance. These results make H2A.Z a potential candidate as a diagnostic biomarker and therapeutic target for PDAC.
Assuntos
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , beta-Galactosidase/genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Envelhecimento/genética , Animais , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Transformação Celular Neoplásica/genética , Dano ao DNA/efeitos dos fármacos , Metilação de DNA/genética , Reparo do DNA/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética/genética , Xenoenxertos , Histonas/genética , Humanos , Camundongos , GencitabinaRESUMO
Hyper-IgE syndrome (HIES) is an immunodeficiency disorder that is characterized by distinctive immunologic and non-immunologic manifestations. Although mutations in signal transducer and activator of transcription 3 (STAT3) have been associated with HIES, the exact nature of the relationship is unknown. Here, we characterized the functional activity of STAT3 and its mutations in 11 Mexican patients with autosomal dominant HIES. STAT3 phosphorylation was evaluated by flow cytometry, and in silico analyses were performed to estimate the impact of allelic mutations on the DNA binding and SH2 domains of the STAT3 protein. Electrophoretic mobility shift assays were used to assess whether the STAT3 mutants could bind to the consensus oligonucleotide target in vitro. Two novel mutations [g.58891A>T (Asn395Tyr) and g.59078A>T (Asn425Tyr)] as well as one possible somatic mosaicism were found in several of the patients who bore some remarkable features. However, there were no direct correlations between genotypes and HIES clinical features. STAT3 phosphorylation was found to be lower in the patient cohort than in healthy controls. Moreover, the mutated STAT3 proteins could bind to the Sp1, but not to the STAT3, consensus sequence. From these functional studies, the STAT3 mutations found in our patient cohort were concluded to be deleterious for normal STAT3 function.
Assuntos
Síndrome de Job/genética , Mutação/genética , Fator de Transcrição STAT3/genética , Sequência de Aminoácidos , Sequência de Bases , Estudos de Coortes , Sequência Consenso , Demografia , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Heterogeneidade Genética , Humanos , Masculino , México , Fosforilação , Fosfotirosina/metabolismo , Ligação Proteica , Fator de Transcrição STAT3/químicaRESUMO
The actin cytoskeleton consists of multiple actin binding proteins (ABPs) that participate cooperatively in different cellular functions such as the maintenance of polarity and cell motility as well as the invasion of target cells and regulation of gene expression, among others. Due to the important role of ABPs in the pathogenesis of Entamoeba histolytica, the role of a new nucleocytoplasmic ABP from E. histolytica named EhNCABP166 was investigated. The EhNCABP166 gene encodes a protein with an estimated molecular weight of 166kDa. Structurally, this peptide is composed of two CH domains arranged in tandem at the N-terminus of the protein, followed by an alpha-helical region containing a number of different domains with a low level of homology. Two (Bin1/Amphiphysin/Rvs167) (BAR) domains, one GTPase-binding/formin 3 homology (GBD/FH3) domain, three Bcl2-associated athanogene (BAG) domains, one basic-leucine zipper (bZIP) domain and one poly(A)-binding protein C-terminal (PABC) domain were also present. Molecular and biochemical studies showed that the EhNCABP166 protein is transcribed and translated in trophozoites of E. histolytica. It was also shown that the CH domains are functional and bind to F-actin, whereas the BAR and GBD/FH3 domains interact in vitro and in vivo with different families of GTPases such as Rho and Ras, and with different phosphoinositides. These findings suggest that these domains have the conserved functional properties described in other eukaryotic systems. These domains also interacted with additional GTPase and lipid targets that have not been previously described. Finally, cellular studies showed that EhNCABP166 is localized to the cytoplasm and nucleus of E. histolytica and that it has an important role in phagocytosis, proliferation, and motility of E. histolytica.
Assuntos
Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Núcleo Celular/química , Citoplasma/química , Entamoeba histolytica/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Perfilação da Expressão Gênica , Metabolismo dos Lipídeos , Proteínas dos Microfilamentos/biossíntese , Proteínas dos Microfilamentos/química , Dados de Sequência Molecular , Peso Molecular , Fagocitose , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
This paper reports the EhGEF1-EhRacG and EhGEF1-EhRho1 molecular complexes from Entamoeba histolytica. The not conserved amino acids Gln201,Tyr299, Gln302, Lys312, Asn313, Phe314 and Ile324 were localized, by means of an in silico computational analysis, at the interface of the exposed face from the DH domain of EhGEF1, which are important to establish the contact with its target GTPases EhRacG and EhRho1. Functional studies of nucleotide exchange of Phe314Ala mutant showed a decrement of 80% on EhRacG GTPase; in contrast the Ile324Ala mutant exhibited a reduction of 77%, specifically on EhRho1; meanwhile the Gln302Ala mutant showed a reduction of approximately 50% on the exchange activity for both GTPases. Moreover, the functional studies of the protein EhGEF1 mutants in the conserved residues Thr194Ala, Asn366Ala and Glu367Ala indicated that contrary to what has been reported for other systems, the mutation of these residues did not alter considerably its catalytic activity.
Assuntos
Canais de Cloreto/química , Canais de Cloreto/genética , Entamoeba histolytica/fisiologia , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Canais de Cloreto/metabolismo , Análise Mutacional de DNA , Entamoeba histolytica/genética , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Quaternária de Proteína , Proteínas de Protozoários/metabolismoRESUMO
Many protozoan parasites represent an important group of human pathogens. Pulsed Field Gradient Gel Electrophoresis (PFGE) analysis has been an important tool for fundamental genetic studies of parasites like Trypanosoma, Leishmania, Giardia or the human malaria parasite Plasmodium falciparum. We present PFGE conditions allowing a high resolution separation of chromosomes ranging from 500 to 4000 kb within a two day electrophoresis run. In addition, we present conditions for separating large chromosomes (2000-6000 kb) within 36 hr. We demonstrate that the application of two dimensional PFGE (2D-PFGE) technique to parasite karyotypes is a very useful method for the analysis of dispersed gene families and comparative studies of the intrachromosomal genome organization.
Assuntos
Mapeamento Cromossômico , Eucariotos/genética , Animais , Eletroforese em Gel de Campo Pulsado , Eucariotos/parasitologia , Plasmodium falciparum/genéticaRESUMO
Our research work group has been interested in the study of the ribosomal RNA and 5S gene systems from Trypanosoma cruzi. Our contributions span from the general description of a multifragmented molecular system, to the sequence analysis of some ribosomal RNA coding regions. From the latter, we have constructed phylogenetic trees of the Trypanosomatidae family, and our data indicate that the molecular inferences do not sustain the traditional classification of these species. Our published findings are here reviewed along with recent unpublished observations of ribosomal RNA and 5S gene structures.