RESUMO
We previously reported a multigene family of monodomain Kunitz proteins from Echinococcus granulosus (EgKU-1-EgKU-8), and provided evidence that some EgKUs are secreted by larval worms to the host interface. In addition, functional studies and homology modeling suggested that, similar to monodomain Kunitz families present in animal venoms, the E. granulosus family could include peptidase inhibitors as well as channel blockers. Using enzyme kinetics and whole-cell patch-clamp, we now demonstrate that the EgKUs are indeed functionally diverse. In fact, most of them behaved as high affinity inhibitors of either chymotrypsin (EgKU-2-EgKU-3) or trypsin (EgKU-5-EgKU-8). In contrast, the close paralogs EgKU-1 and EgKU-4 blocked voltage-dependent potassium channels (Kv); and also pH-dependent sodium channels (ASICs), while showing null (EgKU-1) or marginal (EgKU-4) peptidase inhibitory activity. We also confirmed the presence of EgKUs in secretions from other parasite stages, notably from adult worms and metacestodes. Interestingly, data from genome projects reveal that at least eight additional monodomain Kunitz proteins are encoded in the genome; that particular EgKUs are up-regulated in various stages; and that analogous Kunitz families exist in other medically important cestodes, but not in trematodes. Members of this expanded family of secreted cestode proteins thus have the potential to block, through high affinity interactions, the function of host counterparts (either peptidases or cation channels) and contribute to the establishment and persistence of infection. From a more general perspective, our results confirm that multigene families of Kunitz inhibitors from parasite secretions and animal venoms display a similar functional diversity and thus, that host-parasite co-evolution may also drive the emergence of a new function associated with the Kunitz scaffold.
Assuntos
Equinococose/metabolismo , Equinococose/parasitologia , Proteínas de Helminto/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Inibidores de Serina Proteinase/fisiologia , Animais , Echinococcus granulosus , Gânglios Espinais/efeitos dos fármacos , Modelos Moleculares , Técnicas de Patch-Clamp , Filogenia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , Ratos , Ratos Wistar , Inibidores de Serina Proteinase/farmacologia , Canais de Sódio Disparados por Voltagem/efeitos dos fármacosRESUMO
Serpins are a structurally conserved family of macromolecular inhibitors found in numerous biological systems. The completion and annotation of the genomes of Schistosoma mansoni and Schistosoma japonicum has enabled the identification by phylogenetic analysis of two major serpin clades. S. mansoni shows a greater multiplicity of serpin genes, perhaps reflecting adaptation to infection of a human host. Putative targets of schistosome serpins can be predicted from the sequence of the reactive center loop (RCL). Schistosome serpins may play important roles in both post-translational regulation of schistosome-derived proteases, as well as parasite defense mechanisms against the action of host proteases.
Assuntos
Schistosoma japonicum/enzimologia , Schistosoma mansoni/enzimologia , Inibidores de Serina Proteinase/genética , Serpinas/genética , Animais , Homeostase , Humanos , Filogenia , Schistosoma japonicum/patogenicidade , Schistosoma mansoni/patogenicidade , Esquistossomose/parasitologia , Inibidores de Serina Proteinase/fisiologia , Serpinas/fisiologiaRESUMO
Serpins are a structurally conserved family of macromolecular inhibitors found in numerous biological systems. The completion and annotation of the genomes of Schistosoma mansoni and Schistosoma japonicum has enabled the identification by phylogenetic analysis of two major serpin clades. S. mansoni shows a greater multiplicity of serpin genes, perhaps reflecting adaptation to infection of a human host. Putative targets of schistosome serpins can be predicted from the sequence of the reactive center loop (RCL). Schistosome serpins may play important roles in both post-translational regulation of schistosome-derived proteases, as well as parasite defense mechanisms against the action of host proteases.
Serpinas são uma família de inibidores macromoleculares estruturalmente conservados encontrados em inúmeros sistemas biológicos. O término e a anotação dos genomas de Schistosoma mansoni e de Schistosoma japonicum permitiram a identificação por análise filogenética de dois principais clados de serpinas. S. mansoni mostra uma multiplicidade maior de genes de serpinas, talvez refletindo uma adaptação à infecção de um hospedeiro humano. Alvos putativos das serpinas de esquistossomos podem ser preditos a partir da sequência do "loop" do centro reativo. Serpinas de esquistossomos podem ter importantes papeis tanto na regulação pós-traducional de proteases derivadas do esquistossoma, quanto nos mecanismos de defesa contra a ação de proteases do hospedeiro.
Assuntos
Animais , Humanos , Schistosoma japonicum/enzimologia , Schistosoma mansoni/enzimologia , Inibidores de Serina Proteinase/genética , Serpinas/genética , Homeostase , Filogenia , Schistosoma japonicum/patogenicidade , Schistosoma mansoni/patogenicidade , Esquistossomose/parasitologia , Inibidores de Serina Proteinase/fisiologia , Serpinas/fisiologiaRESUMO
Leishmania major is a protozoan parasite that causes skin ulcerations in cutaneous leishmaniasis. In the mammalian host, the parasite resides in professional phagocytes and has evolved to avoid killing by macrophages. We identified L. major genes encoding inhibitors of serine peptidases (ISPs), which are orthologs of bacterial ecotins, and found that ISP2 inhibits trypsin-fold S1A family peptidases. In this study, we show that L. major mutants deficient in ISP2 and ISP3 (Δisp2/3) trigger higher phagocytosis by macrophages through a combined action of the complement type 3 receptor, TLR4, and unregulated activity of neutrophil elastase (NE), leading to parasite killing. Whereas all three components are required to mediate enhanced parasite uptake, only TLR4 and NE are necessary to promote parasite killing postinfection. We found that the production of superoxide by macrophages in the absence of ISP2 is the main mechanism controlling the intracellular infection. Furthermore, we show that NE modulates macrophage infection in vivo, and that the lack of ISP leads to reduced parasite burdens at later stages of the infection. Our findings support the hypothesis that ISPs function to prevent the activation of TLR4 by NE during the Leishmania-macrophage interaction to promote parasite survival and growth.
Assuntos
Líquido Intracelular/parasitologia , Leishmania major/enzimologia , Leishmania major/crescimento & desenvolvimento , Elastase de Leucócito/fisiologia , Macrófagos Peritoneais/parasitologia , Inibidores de Serina Proteinase/fisiologia , Serpinas/fisiologia , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/metabolismo , Animais , Células Cultivadas , Interações Hospedeiro-Parasita/imunologia , Líquido Intracelular/enzimologia , Líquido Intracelular/imunologia , Leishmania major/imunologia , Elastase de Leucócito/antagonistas & inibidores , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo/imunologia , Inibidores de Serina Proteinase/deficiência , Inibidores de Serina Proteinase/genética , Serpinas/deficiência , Serpinas/genética , Receptor 4 Toll-Like/deficiênciaRESUMO
Serine proteases play key roles in many biological processes, regulating surface proteins that are key-points in signaling pathways. Several studies have reported the presence of members of this protease family in sperm from various species. The precise regulation of their activity is thought to be performed by specific endogenous or extrinsic inhibitors. The contribution of the sperm serine to proteases to fertilization has been demonstrated by synthetic inhibitors and several single knock out experiments, but to date, there is no evidence that links a single enzyme to a single step of fertilization. The explanation for the failure in the understanding of the "one-enzyme-one-process" hypothesis may be that sperm have multiple serine proteases as a mechanism to ensure the success of fertilization. In addition to the classical purification and expression studies, we summarized recent advances in proteomics and performed a bioinformatics search of proteases and inhibitors, providing support for the idea of redundancy. This review summarizes current knowledge about serine proteases and their inhibitors in sperm capacitation and maturation, identifies questions that need to be answered, and provides a reference for future research.
Assuntos
Fertilização/fisiologia , Mamíferos/fisiologia , Serina Proteases/fisiologia , Espermatozoides/enzimologia , Animais , Masculino , Serina Proteases/metabolismo , Inibidores de Serina Proteinase/fisiologia , Capacitação Espermática , Maturação do EspermaRESUMO
Blood-sucking arthropods are vectors responsible for the transmission of several pathogens and parasites to vertebrate animals. The horn fly Haematobia irritans irritans (Diptera: Muscidae) and the tick Boophilus microplus are important hematophagous ectoparasites that cause losses in cattle production. A serine protease inhibitor from a thorax extract of the fly H. irritans irritans (HiTI) was previously isolated, characterized and cloned. In the present study we described the expression, purification, and characterization of the recombinant HiTI (rHiTI) and its possible role in the control of different endogenous and bacterial proteases. rHiTI was successfully expressed using the pPIC9 expression vector with a yield of 4.2 mg/L of active rHiTI. The recombinant HiTI purified by affinity chromatography on trypsin-Sepharose had a molecular mass of 6.53 kDa as determined by LS-ESI mass spectrometry and inhibition constants (Kis) similar to those of native HiTI for bovine trypsin and human neutrophil elastase of 0.4 and 1.0 nM, respectively. Purified rHiTI also showed inhibitory activity against the trypsin-like enzyme of H. i. irritans using its possible natural substrates, fibrinogen and hemoglobin; and also inhibited the OmpT endoprotease of Escherichia coli using fluorogenic substrates. The present results confirm that HiTI may play a role in the control of fly endogenous proteases but also suggest a role in the inhibition of pathogen proteases.
Assuntos
Muscidae/química , Inibidores de Serina Proteinase/fisiologia , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia de Afinidade , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Fibrinogênio/metabolismo , Regulação Enzimológica da Expressão Gênica , Hemoglobinas/metabolismo , Elastase de Leucócito/antagonistas & inibidores , Dados de Sequência Molecular , Peso Molecular , Muscidae/enzimologia , Muscidae/imunologia , Reação em Cadeia da Polimerase , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/genética , Inibidores de Serina Proteinase/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray , Tripsina/efeitos dos fármacos , Tripsina/metabolismoRESUMO
Heparin Cofactor II (HCII) is a glycoprotein in human plasma which inactivates thrombin rapidly in the presence of dermatan sulfate. Inhibition occurs by formation of a stable equimolar complex between HCII and thrombin. HCII association with thrombotic events has not always been observed, thus decreased HCII does not appear to be a strong risk factor for thromboembolic events. Reduced HCII levels have been detected in different clinical conditions, such as hepatic failure, disseminated intravascular coagulation, thalasemina, sickle cell anemia. Increased physiological levels have been found in pregnant women and oral contraception. In our laboratory, we measured HCII plasmatic levels in the normal Buenos Aires city population and in patients under different clinical conditions, such as sepsis, diabetis, burns, oral anticoagulation and in patients treated with heparin, hyperhomcysteinemia in whom septic and diabetic patients showed decreased values. HCII thrombin inhibition possibly takes place in extravascular sites where dermatan sulfate is present. HCII activity would be important in the regulation of wound healing, inflammation, or neuronal development.
Assuntos
Cofator II da Heparina/fisiologia , Inibidores de Serina Proteinase/fisiologia , Trombina/antagonistas & inibidores , Transtornos de Proteínas de Coagulação , Dermatan Sulfato/fisiologia , Cofator II da Heparina/química , Cofator II da Heparina/deficiência , Humanos , Valores de ReferênciaRESUMO
El Cofactor II de la Heparina (HCII) es una proteína perteneciente al sistema de coagulación que inhibe específicamente trombina, processo que es potenciado por acción de los glicosaminoglicanos dermatán sulfato y heparina. Hasta el momento las deficiencias congénitas de HCII encontradas en forma aislada no están asociadas con eventos trombóticos, sí desarollan eventos trombóticos cuando están asociadas a otros factores predisponentes. Se observó disminución en los niveles de HCII en hepatopatías, coagulación intravascular diseminada, en anemia drepanocítica, encontrándose niveles elevados en embarazo a término y por el uso de contraceptivos orales. En el laboratorio realizamos el dosaje del HCII en la población normal de la ciudad de Buenos Aires, en diversas patologías como sepsis, quemados , anticoagulados con dicumarínicos y con heparina, diabéticos, hiperhomocisteinemia, observándose valores disminuidos principalmente en sepsis y pacientes diabéticos. El HCII es una glicoproteína que participa en la inhibición de trombina pero cuyo rol fisiológico no está completamente esclarecido. Es probable que el HCII inhiba trombina en el espacio extravascular, y está relacionado con la regulación de procesos inflamatorios y de reparación tisular.
Assuntos
Humanos , Cofator II da Heparina/fisiologia , Inibidores de Serina Proteinase/fisiologia , Trombina , Trombina/antagonistas & inibidores , Transtornos de Proteínas de Coagulação , Dermatan Sulfato/fisiologia , Cofator II da Heparina/química , Cofator II da Heparina/deficiência , Valores de Referência , RiscoRESUMO
El Cofactor II de la Heparina (HCII) es una proteína perteneciente al sistema de coagulación que inhibe específicamente trombina, processo que es potenciado por acción de los glicosaminoglicanos dermatán sulfato y heparina. Hasta el momento las deficiencias congénitas de HCII encontradas en forma aislada no están asociadas con eventos trombóticos, sí desarollan eventos trombóticos cuando están asociadas a otros factores predisponentes. Se observó disminución en los niveles de HCII en hepatopatías, coagulación intravascular diseminada, en anemia drepanocítica, encontrándose niveles elevados en embarazo a término y por el uso de contraceptivos orales. En el laboratorio realizamos el dosaje del HCII en la población normal de la ciudad de Buenos Aires, en diversas patologías como sepsis, quemados , anticoagulados con dicumarínicos y con heparina, diabéticos, hiperhomocisteinemia, observándose valores disminuidos principalmente en sepsis y pacientes diabéticos. El HCII es una glicoproteína que participa en la inhibición de trombina pero cuyo rol fisiológico no está completamente esclarecido. Es probable que el HCII inhiba trombina en el espacio extravascular, y está relacionado con la regulación de procesos inflamatorios y de reparación tisular. (AU)