Assuntos
Surtos de Doenças/prevenção & controle , Desenho de Fármacos , Vacinas contra Influenza , Influenza Humana/epidemiologia , Tecnologia Farmacêutica , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Humanos , Vacinas contra Influenza/classificação , Vacinas contra Influenza/economia , Vacinas contra Influenza/normas , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Marketing , Vigilância de Produtos Comercializados , Tecnologia Farmacêutica/economia , Tecnologia Farmacêutica/normasRESUMO
The mitogen-activated protein (MAP) kinases are a group of serine/threonine kinases that mediate intracellular signal transduction in response to environmental stimuli including stress, growth factors, and various cytokines. Of this family, the c-Jun N-terminal kinases (JNKs) are members which, depending on cell type, have been shown to activate the transcription of genes involved in the inflammatory response, apoptosis, and hypertrophy. Here we report the use Baculovirus/Sf9 cells to produce milligram quantities of recombinant JNK2beta2 substrate which could be purified to >90% as judged by SDS-PAGE. In addition, we report a novel method for the site-specific biotinylation for this enzyme and demonstrate that the biotinylated product is an authentic substrate of the upstream kinases MKK4 and 7 and can phosphorylate a downstream target, ATF-2. We also show that the phosphorylated product can be captured efficiently on streptavidin-coated beads for use in scintillation proximity assays.
Assuntos
Clonagem Molecular/métodos , Proteínas Quinases Ativadas por Mitógeno/genética , Sequência de Aminoácidos , Baculoviridae , Biotinilação , Linhagem Celular , Escherichia coli , Expressão Gênica , Proteína Quinase 9 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/isolamento & purificação , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The inhibitor-of-apoptosis proteins (IAPs) play a critical role in the regulation of apoptosis by binding and inhibiting caspases. Reaper family proteins and Smac/DIABLO use a conserved amino-terminal sequence to bind to IAPs in flies and mammals, respectively, blocking their ability to inhibit caspases and thus promoting apoptosis. Here we have identified the serine protease Omi/HtrA2 as a second mammalian XIAP-binding protein with a Reaper-like motif. This protease autoprocesses to form a protein with amino-terminal homology to Smac/DIABLO and Reaper family proteins. Full-length Omi/HtrA2 is localized to mitochondria but fails to interact with XIAP. Mitochondria also contain processed Omi/HtrA2, which, following apoptotic insult, translocates to the cytosol, where it interacts with XIAP. Overexpression of Omi/HtrA2 sensitizes cells to apoptosis, and its removal by RNA interference reduces cell death. Omi/HtrA2 thus extends the set of mammalian proteins with Reaper-like function that are released from the mitochondria during apoptosis.