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1.
Mol Cell Biol ; 22(13): 4544-55, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12052864

RESUMO

A variety of cellular stresses activate the stress-responsive mitogen-activated protein (MAP) kinases p38 and JNK. In this study, we studied the activation mechanism of a human MAP kinase kinase kinase, MTK1 (also known as MEKK4), which mediates activation of both p38 and JNK. MTK1 has an extensive N-terminal noncatalytic domain composed of approximately 1,300 amino acids. Full-length or near full-length MTK1 is catalytically inactive when expressed in Saccharomyces cerevisiae cells, as it is in mammalian cells. Deletion of a segment including positions 253 to 553 activates kinase, indicating that this segment contains the autoinhibitory domain. In the autoinhibited conformation, the MTK1 kinase domain cannot interact with its substrate, MKK6. By a functional complementation screening with yeast cells, GADD45 proteins (GADD45alpha, beta, and gamma) were identified as MTK1 activators. GADD45 proteins bind a site in MTK1 near the inhibitory domain and relieve autoinhibition. Mutants of full-length MTK1 were isolated that can interact with MKK6 in the absence of the activator GADD45 proteins. These MTK1 mutants are constitutively active, in both yeast and mammalian cells. A model of MTK1 autoinhibition by the N-terminal inhibitory domain and activation by GADD45 binding is presented.


Assuntos
MAP Quinase Quinase Quinases/metabolismo , Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Domínio Catalítico , Teste de Complementação Genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , MAP Quinase Quinase 6 , MAP Quinase Quinase Quinase 4 , MAP Quinase Quinase Quinases/genética , Mutação , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Proteínas/genética , Saccharomyces cerevisiae/genética , Proteínas GADD45
2.
Vaccine ; 27(8): 1154-65, 2009 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-19146908

RESUMO

The renewed interest in strategies to combat infectious agents with epidemic potential has led to a re-examination of vaccination protocols against smallpox. To help define which antigens elicit a human antibody response, we have targeted proteins known or predicted to be presented on the surface of the intracellular mature virion (IMV) or the extracellular enveloped virion (EEV). The predicted ectodomains were expressed in a mammalian in vitro coupled transcription/translation reaction using tRNA(lys) precharged with lysine-epsilon-biotin followed by solid phase immobilization on 384-well neutravidin-coated plates. The generated array is highly specific and sensitive in a micro-ELISA format. By comparison of binding of vaccinia-immune sera to the reticulocyte lysate-produced proteins and to secreted post-translationally modified proteins, we demonstrate that for several proteins including the EEV proteins B5 and A33, proper recognition is dependent upon appropriate folding, with little dependence upon glycosylation per se. We further demonstrate that the humoral immune response to vaccinia among different individuals is not uniform in specificity or strength, as different IMV and EEV targets predominate within the group of immunogenic proteins. This heterogeneity likely results from the diversity of HLA Class II alleles and CD4 T helper cell epitopes stimulating B cell antibody production. Our findings have important implications both for design of new recombinant subunit vaccines as well as for methods of assaying the human antibody response utilizing recombinant proteins produced in vitro.


Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Análise Serial de Proteínas , Vacina Antivariólica/imunologia , Vaccinia virus/imunologia , Variação Genética , Humanos , Testes de Neutralização , Proteínas Estruturais Virais/imunologia
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