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Mol Cell Biol ; 25(3): 1089-99, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15657435


Animal cells counteract oxidative stress and electrophilic attack through coordinated expression of a set of detoxifying and antioxidant enzyme genes mediated by transcription factor Nrf2. In unstressed cells, Nrf2 appears to be sequestered in the cytoplasm via association with an inhibitor protein, Keap1. Here, by using the yeast two-hybrid screen, human Keap1 has been identified as a partner of the nuclear protein prothymosin alpha. The in vivo and in vitro data indicated that the prothymosin alpha-Keap1 interaction is direct, highly specific, and functionally relevant. Furthermore, we showed that Keap1 is a nuclear-cytoplasmic shuttling protein equipped with a nuclear export signal that is important for its inhibitory action. Prothymosin alpha was able to liberate Nrf2 from the Nrf2-Keap1 inhibitory complex in vitro through competition with Nrf2 for binding to the same domain of Keap1. In vivo, the level of Nrf2-dependent transcription was correlated with the intracellular level of prothymosin alpha by using prothymosin alpha overproduction and mRNA interference approaches. Our data attribute to prothymosin alpha the role of intranuclear dissociator of the Nrf2-Keap1 complex, thus revealing a novel function for prothymosin alpha and adding a new dimension to the molecular mechanisms underlying expression of oxidative stress-protecting genes.

Proteínas de Ligação a DNA/metabolismo , Estresse Oxidativo/genética , Precursores de Proteínas/metabolismo , Proteínas/metabolismo , Timosina/análogos & derivados , Timosina/metabolismo , Transativadores/metabolismo , Ativação Transcricional/genética , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteína 1 Associada a ECH Semelhante a Kelch , Fator 2 Relacionado a NF-E2 , Estresse Oxidativo/fisiologia , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Ativação Transcricional/fisiologia , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido
Exp Cell Res ; 284(2): 211-23, 2003 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-12651154


Human prothymosin alpha is a proliferation-related nuclear protein undergoing caspase-mediated fragmentation in apoptotic cells. We show here that caspase-3 is the principal executor of prothymosin alpha fragmentation in vivo. In apoptotic HeLa cells as well as in vitro, caspase-3 cleaves prothymosin alpha at one major carboxy terminal (DDVD(99)) and several suboptimal sites. Prothymosin alpha cleavage at two amino-terminal sites (AAVD(6) and NGRD(31)) contributes significantly to the final pattern of prothymosin alpha fragmentation in vitro and could be detected to occur in apoptotic cells. The major caspase cleavage at D(99) disrupts the nuclear localization signal of prothymosin alpha, which leads to a profound alteration in subcellular localization of the truncated protein. By using a set of anti-prothymosin alpha monoclonal antibodies, we were able to observe nuclear escape and cell surface exposure of endogenous prothymosin alpha in apoptotic, but not in normal, cells. We demonstrate also that ectopic production of human prothymosin alpha and its mutants with nuclear or nuclear-cytoplasmic localization confers increased resistance of HeLa cells toward the tumor necrosis factor-induced apoptosis.

Apoptose/fisiologia , Caspases/metabolismo , Células Eucarióticas/metabolismo , Fragmentos de Peptídeos/metabolismo , Precursores de Proteínas/biossíntese , Transporte Proteico/fisiologia , Timosina/análogos & derivados , Timosina/biossíntese , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Sequência de Aminoácidos/fisiologia , Anticorpos Monoclonais , Apoptose/efeitos dos fármacos , Caspase 3 , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Células HeLa , Humanos , Mutação/genética , Precursores de Proteínas/antagonistas & inibidores , Precursores de Proteínas/genética , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/efeitos dos fármacos , Timosina/antagonistas & inibidores , Timosina/genética
J Immunol Methods ; 266(1-2): 185-96, 2002 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-12133636


To overcome poor immunogenicity of prothymosin alpha, a small and highly acidic nuclear protein involved in cell proliferation, production of anti-prothymosin alpha antibodies in mice immunized with free human prothymosin alpha, with prothymosin alpha coupled to different carriers and with prothymosin alpha fused to green fluorescent protein was assessed. Fusing prothymosin alpha to green fluorescent protein turned out to be the superior approach resulting in production of high titer anti-prothymosin alpha antibodies. From these studies, two highly specific anti-prothymosin alpha monoclonal antibodies recognizing epitopes within the amino terminal (2F11) and middle (4F4) portions of the human prothymosin alpha molecule were obtained and characterized. As expected, the 2F11 antibody displayed broad species specificity, whereas the 4F4 antibody appeared to be species-specific permitting discrimination of human versus rat protein. Furthermore, a combination of point mutations in prothymosin alpha that alter the properties of the protein precluded recognition by the 4F4 antibody. Intramolecular masking of the 4F4 epitope in prothymosin alpha fused to the Tat transduction peptide of human immunodeficiency virus type 1 was observed. The anti-prothymosin alpha antibodies obtained were suitable for precipitation of human prothymosin alpha from HeLa cell lysates and for immunolocalization of the endogenous prothymosin alpha within the cells. Fusion with green fluorescent protein may thus be helpful in raising antibodies against 'problematic' proteins.

Anticorpos Monoclonais/imunologia , Precursores de Proteínas/imunologia , Timosina/análogos & derivados , Timosina/imunologia , Animais , Especificidade de Anticorpos , Mapeamento de Epitopos , Produtos do Gene tat/genética , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos BALB C , Mutação Puntual , Conformação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusão/imunologia , Especificidade da Espécie , Timosina/química , Timosina/genética