Assuntos
Cisteína Endopeptidases/química , Precursores Enzimáticos/química , Proteínas de Plantas/química , Estrutura Secundária de Proteína , Clorometilcetonas de Aminoácidos/química , Sequência de Aminoácidos , Catepsina B/química , Simulação por Computador , Cristalografia por Raios X , Cisteína Endopeptidases/genética , Inibidores de Cisteína Proteinase , Ativação Enzimática , Precursores Enzimáticos/genética , Frutas , Ligação de Hidrogênio , Leucina/análogos & derivados , Leucina/química , Leupeptinas/química , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Oligopeptídeos/química , Papaína/química , Proteínas de Plantas/genética , Processamento de Proteína Pós-Traducional , Homologia de Sequência de AminoácidosRESUMO
In this study, we showed that plasminogen (Plg) and plasmin (Pla) bind to lysine-binding sites on cell surface and trigger a signaling pathway that activates the mitogen-activated protein kinase (MAPK) MEK and ERK1/2, which in turn leads to the expression of the primary response genes c-fos and early growth response gene egr-1. Our data show that the Plg/Pla-stimulated steady-state mRNA levels of both genes reached a maximum by 30 min and then returned to basal levels by 1h. The gene induction was sensitive to both pharmacological and genetic inhibition of MEK. Leupeptin, a serine protease inhibitor, suppressed Pla but not Plg-induced c-fos and egr-1 expression, emphasizing the role played by the serine protease activity associated with Pla. Pre-incubation with cholera toxin completely blocked the Plg/Pla-induced gene expression, suggesting that another signaling pathway, which recruits G protein-coupled receptors, may also be involved. Furthermore, Plg/Pla also stimulated AP-1 and EGR-1 DNA-binding activities, which were abrogated by pharmacological inhibition of MEK. Altogether, these results suggest that Plg/Pla stimulates c-fos and egr-1 expression via activation of the MEK/ERK pathway.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Fibrinolisina/fisiologia , Proteínas Imediatamente Precoces/biossíntese , Sistema de Sinalização das MAP Quinases , Plasminogênio/fisiologia , Proteínas Proto-Oncogênicas c-fos/biossíntese , Fatores de Transcrição/biossíntese , Animais , Sítios de Ligação , Northern Blotting , Western Blotting , Linhagem Celular , Toxina da Cólera/química , DNA/metabolismo , Relação Dose-Resposta a Droga , Proteína 1 de Resposta de Crescimento Precoce , Ativação Enzimática , Flavonoides/farmacologia , Genes Dominantes , Humanos , Leupeptinas/química , Lisina/química , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Ligação Proteica , RNA/metabolismo , RNA Mensageiro/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
Electrostatic forces are involved in a wide variety of molecular interactions that are of biological interest, including, among others, DNA-Protein interactions, protein folding, and the interactions between enzymes and their substrates and inhibitors. In this work, the interaction between papain and an inhibitor, leupeptin, is analyzed from the point of view of their electrostatic interaction. The computations enable one to suggest that negatively charged amino acids located in the region of the active site are responsible for creating an environment that enables efficient binding of the inhibitor. This binding occurs despite the fact that the net global charge of both molecules is positive; an explanation for this apparent contradiction is proposed.