RESUMO
Leptospirosis is an acute febrile disease caused by pathogenic spirochetes of the genus Leptospira. It is considered an important re-emerging infectious disease that affects humans worldwide. The knowledge about the mechanisms by which pathogenic leptospires invade and colonize the host remains limited since very few virulence factors contributing to the pathogenesis of the disease have been identified. Here, we report the identification and characterization of two new leptospiral proteins with OmpA-like domains. The recombinant proteins, which exhibit extracellular matrix-binding properties, are called Lsa46 - LIC13479 and Lsa77 - LIC10050 (Leptospiral surface adhesins of 46 and 77 kDa, respectively). Attachment of Lsa46 and Lsa77 to laminin was specific, dose dependent and saturable, with KD values of 24.3 ± 17.0 and 53.0 ± 17.5 nM, respectively. Lsa46 and Lsa77 also bind plasma fibronectin, and both adhesins are plasminogen (PLG)-interacting proteins, capable of generating plasmin (PLA) and as such, increase the proteolytic ability of leptospires. The proteins corresponding to Lsa46 and Lsa77 are present in virulent L. interrogans L1-130 and in saprophyte L. biflexa Patoc 1 strains, as detected by immunofluorescence. The adhesins are recognized by human leptospirosis serum samples at the onset and convalescent phases of the disease, suggesting that they are expressed during infection. Taken together, our data could offer valuable information to the understanding of leptospiral pathogenesis.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Leptospira interrogans/genética , Animais , Anticorpos Antibacterianos/sangue , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Genoma Bacteriano , Humanos , Leptospira interrogans/imunologia , Leptospirose/sangue , Leptospirose/imunologia , Leptospirose/microbiologia , Camundongos Endogâmicos BALB C , Filogenia , Plasminogênio/química , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Human tissue kallikreins (KLKs) are a group of serine proteases found in many tissues and biological fluids and are differentially expressed in several specific pathologies. Here, we present evidences of the ability of these enzymes to activate plasminogen. Kallikreins 3 and 5 were able to induce plasmin activity after hydrolyzing plasminogen, and we also verified that plasminogen activation was potentiated in the presence of glycosaminoglycans compared with plasminogen activation by tPA. This finding can shed new light on the plasminogen/plasmin system and its involvement in tumor metastasis, in which kallikreins appear to be upregulated.
Assuntos
Fibrinolisina/química , Calicreínas/química , Inibidor 1 de Ativador de Plasminogênio/química , Plasminogênio/química , Ativador de Plasminogênio Tecidual/química , Sequência de Aminoácidos , Baculoviridae/genética , Compostos Cromogênicos/química , Ensaios Enzimáticos , Humanos , Cinética , Dados de Sequência Molecular , Proteólise , Proteínas Recombinantes/química , SoluçõesRESUMO
Yersinia pestis protein Pla is a plasmid-coded outer membrane protein with aspartic-protease activity. Pla exhibits a plasminogen (Plg) activator activity (PAA) that promotes the cleavage of Plg to the active serine-protease form called plasmin. Exactly how Pla activates Plg into plasmin remains unclear. To investigate this event, we performed the interactions between the predicted Plg and Pla protein structures by rigid-body docking with the HEX program and evaluated the complex stability by molecular dynamics (MD) using the GROMACS package programs. The predicted docked complex of Plg-Pla shows the same interaction site predicted by experimental site-direct mutagenesis in other studies. After a total of 8 ns of MD simulation, we observed the relaxation of the beta-barrel structure of Pla and the progressive approximation and stabilization between the cleavage site of Plg into the extracellular loops of Pla, followed by the increase in the number of H bonds. We also report here the aminoacids that participate in the active site and the sub sites of interaction. The total understanding of these interactions can be an important tool for drug design against bacterial proteases.
Assuntos
Proteínas de Bactérias/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ativadores de Plasminogênio/química , Plasminogênio/química , Yersinia pestis/enzimologia , Sequência de Aminoácidos , Humanos , Ligação de Hidrogênio , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Homologia Estrutural de ProteínaRESUMO
To assess the importance of carbohydrate moieties to the anti-angiogenic activity of plasminogen fragments, we cloned the fragment corresponding to amino acids Val(79) to Thr(346) (Kint3-4) that presents the three glycosylation sites. The activity of glycosylated and unglycosylated Kint3-4 was tested in murine sponge implant model. We observed a significant decrease in the neovascularization on the sponge after treatment with Kint3-4 by histological examination and determination of the hemoglobin levels. The effects were more intense with the glycosylated than the unglycosylated protein. (99m)Technecium-labeled red blood cells confirmed the inhibition of cell infiltration in the implanted sponge. Studies using melanoma B16F1 implanted in a mouse demonstrated that treatment with glycosylated Kint3-4 (0.15 nmol/48 h) during 14 days suppresses tumor growth by 80%. The vascular endothelial growth factor mRNA levels on the tumor were reduced after treatment. Kint3-4 is a potent plasminogen fragment that has been found to inhibit tumor growth.
Assuntos
Inibidores da Angiogênese/farmacologia , Fragmentos de Peptídeos/farmacologia , Plasminogênio/farmacologia , Sequência de Aminoácidos , Animais , Glicosilação , Humanos , Integrina alfaVbeta3/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Plasminogênio/química , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
INTRODUCTION: Dermatan sulfate (DS) is well-known for its anticoagulant activity through binding to heparin cofactor II to enhance antithrombin action. It has also been suggested that DS has a profibrinolytic effect, although the exact molecular mechanism is as yet unknown. MATERIALS AND METHODS: An in vitro amidolytic method was used to study the effect of high and low molecular weight-DS on the activation of Glu and Lys-plasminogen by tissue and urinary plasminogen activators (t-PA and u-PA). RESULTS: Both high and low molecular weight-DS exhibited a stimulating effect on the activation of plasminogen by PAs. Interestingly, high molecular weight-DS stimulated Glu and Lys-plasminogen activation by t-PA and u-PA in a way and to an extent similar to that in which fibrin(ogen) degradation products (PDF) increased the t-PA assay. Meanwhile low molecular weight-DS had a lower effect. No DS had any effect on plasmin or u-PA amidolytic activity. The facilitation of the conversion of Glu-plasminogen to plasmin in the presence of DS was confirmed by SDS-PAGE; high molecular weight-DS effect was greater than low molecular weight-DS in accordance with the chromogenic assays. Moreover, the combination of PDF and high and low molecular weight-DS, respectively, did not further stimulate t-PA activation of either Glu or Lys-plasminogen suggesting that both substances may compete for the same binding sites. CONCLUSIONS: Through in vitro assays we demonstrated that high and low molecular weight-DS enhance plasminogen activation by u-PA and t-PA, suggesting that the profibrinolytic activity of DS might be via potentiation of plasminogen conversion to plasmin.
Assuntos
Dermatan Sulfato/farmacologia , Fibrinólise/efeitos dos fármacos , Anticoagulantes/metabolismo , Antitrombinas/metabolismo , Dermatan Sulfato/química , Fibrinolisina/metabolismo , Ácido Glutâmico/química , Humanos , Hidrólise , Modelos Químicos , Peso Molecular , Fragmentos de Peptídeos/química , Plasminogênio/química , Ativadores de Plasminogênio/metabolismo , Fatores de Tempo , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Human kallikrein 6 (hK6) is abundantly expressed in the central nervous system and is implicated in demyelinating disease. This study provided biochemical data about the substrate specificity and activation of hK6 by glycosaminoglycans and by kosmotropic salts, which followed the Hofmeister series. The screening of fluorescence resonance energy transfer (FRET) peptide families derived from Abz-KLRSSKQ-EDDnp resulted in the finding that Abz-AFRFSQ-EDDnp (where Abz is ortho-aminobenzoic acid and EDDnp is N-[2,4-dinitrophenyl]ethylenediamine)) is the best synthetic substrate described so far for hK6 (kcat/Km 38,667 s(-1) mm(-1)). It is noteworthy that the AFRFS sequence was found as a motif in the amino-terminal domain of seven human ionotropic glutamate receptor subunits. We also examined the hK6 hydrolytic activity on FRET peptides derived from human myelin basic protein, precursor of the Abeta amyloid peptide, reactive center loop of alpha1-antichymotrypsin, plasminogen, and maturation and inactivation cleavage sites of hK6, which were described earlier as natural substrates for hK6. The best substrates were derived from myelin basic protein. The hK6 maturation cleavage site was poorly hydrolyzed, and no evidence was found to support a two-step self-activation process reported previously. Finally, we assayed FRET peptides derived from sequences that span the cleavage sites for activation of protease-activated receptors (PAR) 1-4, and only the substrate with the PAR 2 sequence was hydrolyzed. These results further supported the hypothesis that hK6 expressed in the central nervous system is involved in normal myelin turnover/demyelination processes, but it is unlikely to self-activate. This report also suggested the possible modulation of ionotropic glutamate receptors and activation of PAR 2 by hK6.
Assuntos
Glicosaminoglicanos/química , Calicreínas/química , Sais/química , Amiloide/química , Peptídeos beta-Amiloides/química , Sítios de Ligação , Citratos/química , Relação Dose-Resposta a Droga , Transferência Ressonante de Energia de Fluorescência , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Modelos Químicos , Proteína Básica da Mielina/química , Bainha de Mielina/química , Peptídeos/química , Plasminogênio/química , Ligação Proteica , Receptor PAR-2/metabolismo , Receptores de Ácido Caínico/química , Proteínas Recombinantes/química , Citrato de Sódio , Especificidade por Substrato , alfa 1-Antiquimotripsina/química , Receptor de GluK2 CainatoRESUMO
Angiostatin is a plasminogen-derived anti-angiogenic factor composed of its first four kringle structures. This molecule is generated by proteolytic cleavage of plasminogen by some proteolytic enzymes in vitro. Since venoms of viper snakes are a rich source of both serine- and metalloproteinase, we hypothesized that angiostatin-like polypeptides could be generated during the envenomation after snake bites and play a pathophysiological role in the local tissue damage and regeneration. Our results showed that crude venoms from several species of Bothrops snakes were able to generate angiostatin-like polypeptides and purified metalloproteinases but not serine proteinases from Bothrops jararaca and Bothrops moojeni venoms were responsible for their generation in vitro. The putative plasminogen cleavage sites by the crude venoms and purified proteinases were determined by N-terminal amino acid sequencing of the angiostatin-like molecules. Angiostatin-like peptides derived from human plasminogen digestion by jararhagin, a metalloproteinase isolated from B. jararaca venom, inhibited endothelial cell proliferation in vitro. These results indicate that angiostatin-like molecules can be generated upon snakebite envenomations and may account for the poor and incomplete regenerative response observed in the damaged tissue.