RESUMO
BACKGROUND: Eukaryote initiation factor 2 subunit ß (eIF2ß) plays a crucial role in regulation protein synthesis, which mediates the interaction of eIF2 with mRNA. eIF2ß contains evolutionarily conserved polylysine stretches in amino-terminal region and a zinc finger motif in the carboxy-terminus. METHODS: The gene eIF2ß was cloned under tetracycline transcription control and the polylysine stretches were deleted by site-directed mutagenesis (eIF2ßΔ3K). The plasmid was transfected into HEK 293 TetR cells. These cells were analyzed for their proliferative and translation capacities as well as cell death rate. Experiments were performed using gene reporter assays, western blotting, flow cytometry, cell sorting, cell proliferation assays and confocal immunofluorescence. RESULTS: eIF2ßΔ3K affected negatively the protein synthesis, cell proliferation and cell survival causing G2 cell cycle arrest and increased cell death, acting in a negative dominant manner against the native protein. Polylysine stretches are also essential for eIF2ß translocated from the cytoplasm to the nucleus, accumulating in the nucleolus and eIF2ßΔ3K did not make this translocation. DISCUSSION: eIF2ß is involved in the protein synthesis process and should act in nuclear processes as well. eIF2ßΔ3K reduces cell proliferation and causes cell death. Since translation control is essential for normal cell function and survival, the development of drugs or molecules that inhibit translation has become of great interest in the scenario of proliferative disorders. In conclusion, our results suggest the dominant negative eIF2ßΔ3K as a therapeutic strategy for the treatment of proliferative disorders and that eIF2ß polylysine stretch domains are promising targets for this.
Assuntos
Proliferação de Células/genética , Fator de Iniciação 2B em Eucariotos/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Polilisina/genética , Biossíntese de Proteínas/genética , Deleção de Sequência/genética , Apoptose/genética , Sítios de Ligação , Núcleo Celular/metabolismo , Sobrevivência Celular/genética , Citoplasma/metabolismo , Fator de Iniciação 2B em Eucariotos/metabolismo , Células HEK293 , Humanos , Terapia de Alvo Molecular/métodos , Mutagênese Sítio-Dirigida , Neoplasias/terapia , Ligação Proteica , Transporte Proteico , RNA Mensageiro/metabolismoRESUMO
Southern Brazil is considered an area of low Hepatitis B endemicity, but some areas of higher endemicity have been described in the Southwest of Paraná and Santa Catarina states. The aim of this study was to evaluate viral genotypes circulating throughout Paraná state. PCR amplification and partial sequencing of the S gene was carried out in 228 samples from HBsAg positive candidate blood donors. Samples have been collected in seven different counties (Cascavel, Curitiba, Foz do Iguaçu, Francisco Beltrão, Maringá, Londrina and Paranaguá). The most common HBV genotype in Paraná state was D (82.9%; 189/228), followed by A (14.1%; 32/228). Genotypes F (1.3%; 3/228), C (1.3%; 3/228) and H (0.4%; 1/228) were also found. Distribution of genotypes was different in the studied counties, but genotype D was the most frequent in all of them. In Francisco Beltrão, all studied samples belonged to genotype D. The high prevalence of HBV genotype D in South of Brazil is explained by the intense migration of settlers from Europeans countries. Subgenotypes A1 and A2 were identified circulating in all cities where HBV/A was found. As observed in other areas of Brazil, HBV/A1 is more frequent than the HBV/A2 in Paraná state and its presence was significantly larger in black and mulatto individuals. Genotype C was found only in individuals with Asian ancestry from Londrina and Maringá. Most HBV/F sequences identified in this study were classified as subgenotype F2a that was previously described in Brazil. The sole case of subgenotype F4 was from Foz do Iguaçu city, near to Northern Argentina, where F4 is highly prevalent. The single genotype H sample was from Curitiba. This is the first case of this genotype described in Brazil. Further studies should be carried out to determine if more genotype H samples can be found in other populations from Brazil.
Assuntos
Vírus da Hepatite B/genética , Hepatite B/virologia , Adulto , Análise de Variância , Doadores de Sangue , Brasil/epidemiologia , Doenças Endêmicas , Europa (Continente)/epidemiologia , Feminino , Genótipo , Hepatite B/epidemiologia , Humanos , Masculino , FilogeniaRESUMO
Human T cell lymphotropic virus type II (HTLV-II) is a deltaretrovirus endemic in Indian populations living in Central and South America, among Pygmies tribes from Central Africa, and epidemic among injecting drug users (IDUs) in the United States, Europe, Southeast Asia, and South America. To date only the HTLV-IIa subtype has been demonstrated among Brazilians (Amazon basin Indians, blood donors, and IDUs). We analyzed HTLV-II isolates from 12 individuals living in the urban area of Porto Alegre, Southern Brazil, identified as seropositive for HTLVI/II in a blood donation. The HTLV-II long terminal repeat (LTR) region was sequenced and compared with nucleotide sequences of isolates HTLV-IIa (Mo), HTLV-IIb (NRA) prototypes. Phylogenetic analysis of the LTR region demonstrated that seven new isolates clustered together with American Indians HTLV-IIb isolates, and five new HTLV-IIa isolates clustered within the HTLV-IIa Brazilian subgroup, named the HTLV-IIc subtype. Both HTLV-IIa and IIb seem to be endemic in the urban area of Porto Alegre, South of Brazil, and could have reached this region via the Amazon basin and the Pacific Coast ancient human migratory pathways. To our knowledge this is the first study demonstrating the presence of HTLV-IIb among the urban population in Brazil.
Assuntos
Infecções por HTLV-II/epidemiologia , Infecções por HTLV-II/virologia , Vírus Linfotrópico T Tipo 2 Humano/genética , Epidemiologia Molecular , Sequência de Bases , Doadores de Sangue/estatística & dados numéricos , Western Blotting , Brasil/epidemiologia , Emigração e Imigração , Ensaio de Imunoadsorção Enzimática , Evolução Molecular , Feminino , Variação Genética , Vírus Linfotrópico T Tipo 2 Humano/classificação , Vírus Linfotrópico T Tipo 2 Humano/isolamento & purificação , Humanos , Masculino , Dados de Sequência Molecular , Técnicas de Amplificação de Ácido Nucleico , Filogenia , Reação em Cadeia da Polimerase , RNA Viral/análise , Análise de Sequência de RNA , Estudos Soroepidemiológicos , Sequências Repetidas Terminais , População UrbanaRESUMO
elF2 está envolvido na seleção do codon AUG para inicio de tradução em eucariotos, uma vez que em levedura, mutações em suas três subunidades foram identificadas por permitirem o reconhecimento de um codon UUG pelo anti-codon do Met-tRNAiMet. A subunidade beta de elF2 contém um motivo que poderia mediar interações com RNA compreendido por três blocos de sete resíduos de lisina, os quais são altamente conservados evolutivamente. Neste trabalho foi realizada uma análise de deleções nesta proteína em Saccharomyces cerevisiae, a qual indicou que somente um bloco de lislnas é suficiente para manter sua função "in vivo", provavelmente devido à carga, uma vez que a troca destes aminoácidos por arglnina, mas não por alanina, manteve a viabilidade celular. A remoção de qualquer um dos bloco de lisinas não afeta a expressão de GCN4. Entre as duplas deleções, somente aquela em que permanece o segundo bloco levou a uma marcada desrepressão de GCN4. A tripla deleção resultou em altos níveis de expressão de GCN4, correspondendo a um fenótipo dominante negativo, indicando que este alelo pode ainda estar mantendo uma função parcial, competindo com a proteína selvagem e proporcionando um defeito na síntese protélca. Esta proteína deletada associa-se com elF2a e elF2g e é encontrada no complexo de pré-iniciação. A ligação de mRNA "in vitro" a proteínas de fusão GST-elF2b é dependente dos blocos de lisinas, sendo mantlda na presença de um único bloco de lisinas ou argininas, mas não de alaninas. A ligação de elF2 ao mRNA é dependente da presença dos blocos de lisinas na subunidadeb. A ligação de elF2 a Met-tRNAiMet de forma GTP-dependente não é afetada pela ausência das repetições de lisinas. Os resultados obtidos aqui fornecem fortes evidências do papel dos blocos de lisinas de elF2b em manter a interação com mRNA "in vivo"