RESUMO
p21-activated kinases (PAKs) are key regulators of actin dynamics, cell proliferation and cell survival. Deregulation of PAK activity contributes to the pathogenesis of various human diseases, including cancer and neurological disorders. Using an ELISA-based screening protocol, we identified naphtho(hydro)quinone-based small molecules that allosterically inhibit PAK activity. These molecules interfere with the interactions between the p21-binding domain (PBD) of PAK1 and Rho GTPases by binding to the PBD. Importantly, they inhibit the activity of full-length PAKs and are selective for PAK1 and PAK3 in vitro and in living cells. These compounds may potentially be useful for determining the details of the PAK signaling pathway and may also be used as lead molecules in the development of more selective and potent PAK inhibitors.
Assuntos
Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/farmacologia , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/metabolismo , Regulação Alostérica/efeitos dos fármacos , Linhagem Celular , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Ativação Enzimática/efeitos dos fármacos , Humanos , Isoenzimas , Ligação Proteica , Inibidores de Proteínas Quinases/química , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/químicaRESUMO
Cell migration requires the coordinated spatiotemporal regulation of actomyosin contraction and cell protrusion/adhesion. Nonmuscle myosin II (MII) controls Rac1 and Cdc42 activation, and cell protrusion and focal complex formation in migrating cells. However, these mechanisms are poorly understood. Here, we show that MII interacts specifically with multiple Dbl family guanine nucleotide exchange factors (GEFs). Binding is mediated by the conserved tandem Dbl homology-pleckstrin homology module, the catalytic site of these GEFs, with dissociation constants of approximately 0.3 microM. Binding to the GEFs required assembly of the MII into filaments and actin-stimulated ATPase activity. Binding of MII suppressed GEF activity. Accordingly, inhibition of MII ATPase activity caused release of GEFs and activation of Rho GTPases. Depletion of betaPIX GEF in migrating NIH3T3 fibroblasts suppressed lamellipodial protrusions and focal complex formation induced by MII inhibition. The results elucidate a functional link between MII and Rac1/Cdc42 GTPases, which may regulate protrusion/adhesion dynamics in migrating cells.
Assuntos
Movimento Celular/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Miosina Tipo II/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Actomiosina/metabolismo , Animais , Sítios de Ligação , Adesão Celular , Ativação Enzimática , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Células Jurkat , Camundongos , Miosina Tipo II/genética , Células NIH 3T3 , Fator de Crescimento Derivado de Plaquetas/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Angiogenin is one of the most potent angiogenesis-inducing proteins. Angiostatin is one of the most potent angiogenesis inhibitors, and it contains the first four kringle domains of plasminogen (K1-4). Recombinant human plasminogen kringle 1-3 (rK1-3) was expressed in Escherichia coli and purified to homogeneity. The binding of t-4-aminomethylcyclohexanecarboxylic acid with the purified kringle 1-3 was determined by changes in intrinsic fluorescence. rK1-3 exhibits comparable ligand-binding properties as native human plasminogen kringle 1-3. The purified rK1-3 inhibits neovascularization in the chick embryo chorioallantoic membrane (CAM) assay. Interaction of angiogenin with rK1-3 was examined by immunological binding assay and surface plasmon resonance kinetic analysis, and the equilibrium dissociation constants for the complex, Kd, are 0.89 and 0.18 microM, respectively. rK1-3 inhibits angiogenin-induced angiogenesis in the chick embryo CAM in a concentration-dependent manner. These results indicate that rK1-3 directly binds to angiogenin and thus rK1-3 inhibits the angiogenic activity of angiogenin.
Assuntos
Inibidores da Angiogênese/metabolismo , Membrana Corioalantoide/irrigação sanguínea , Neovascularização Fisiológica , Fragmentos de Peptídeos/metabolismo , Plasminogênio/metabolismo , Ribonuclease Pancreático/antagonistas & inibidores , Inibidores da Angiogênese/isolamento & purificação , Animais , Embrião de Galinha , Ensaio de Imunoadsorção Enzimática , Ligantes , Fragmentos de Peptídeos/isolamento & purificação , Plasminogênio/isolamento & purificação , Ribonuclease Pancreático/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
A case of tuberculosis involving facial skin, nasal cavity throat and eyes was found in a yong korean girl. We report the case because of its unusual clinical picture and to recall the morbidity of cutaneos tuberculosis in the present dermatological field.Skin tuberculosis is one of the oldest diseases in dermatology. But the invasion of the skin by tubercle bacilli still seen and all the types of so called reinfection tuberculosis of the skin are being found sporadically all over the world.Generally patients with skin tuberculosis adapt themselves to their disorder during the long course of the disease actually, mostof the tuberculous skin lesions do not cause great troubles to the patient allowing him or her to lead a normal life. Recently we found a case which was quite unusual and seriosly treatened the patients normal activity because of naasal obstruction and facial disfiguration.
