Identification and characterization of a novel factor XIIa inhibitor in the hematophagous insect, Triatoma infestans (Hemiptera : Reduviidae)

Recently, we have cloned several Kazal-type serine protease inhibitors from the midgut of the Triatoma infestans bug. A single gene composed of multi Kazal-type domains, in tandem, encodes these inhibitors. in this work, we describe the purification and characterization of recombinant infestins 3-4 and 4, which are potent factor XIIa inhibitors (K-i = 67 pM and 128 pM, respectively). We also identified the first native factor XIIa inhibitor from a hematophagous insect. the factor XIIa inhibitory activity of infestin 4 demonstrates extremely efficient anticoagulant activity, prolonging activated partial thromboplastin time by approximately 3 times. Our results suggest that infestins perform a very important role in the T. infestans midgut during meal acquisition and digestion by controlling blood coagulation by means of inhibiting thrombin and factor XIIa. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.Universidade Federal de São Paulo, Dept Biochem, BR-04044020 São Paulo, BrazilInst Butantan, Physiol Lab, BR-05504900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, BR-04044020 São Paulo, BrazilWeb of Scienc

Characterization of thrombin inhibitory mechanism of rAaTI, a Kazal-type inhibitor from Aedes aegypti with anticoagulant activity

Saliva of blood-sucking arthropods contains a complex mixture of anti-haemostatic, anti-inflammatory and immune-modulator compounds. Among anti-haemostatic factors, there are anticoagulants, vasodilators and platelet aggregation inhibitors. Previous analyses of the sialotranscriptome of Aedes aegypti showed the potential presence of a Kazal-type serine protease inhibitor in the female salivary glands, carcass and also in the whole male, which inhibitor we named AaTI (A. aegypti thrombin inhibitor). Recently, we expressed and characterized rAaTI as a trypsin inhibitor, and its anticoagulant activity [1]. in this work we characterized the thrombin inhibition mechanism of rAaTI. Recombinant AaTI was able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. in contrast, AaTI Delta (rAaTI truncated form) and C-terminal AaTI acidic tail prolong only thrombin time. in the competition assay, rAaTI, AaTI Delta or C-terminal AaTI acidic tail thrombin interactions seem to be affected by heparin but not by hirudin, suggesting that rAaTI binds to thrombin exosite 2. Finally, the thrombin inhibition assay of rAaTI showed an uncompetitive inhibition mechanism. in conclusion, rAaTI can probably inhibit thrombin by interacting with thrombin exosite 2, and the interaction is not mediated by the AaTI C-terminal region, since the truncated AaTI Delta form also prolongs thrombin time. (C) 2010 Elsevier Masson SAS. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilInst Butantan, Lab Fisiopatol, BR-05503900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilFAPESP: 05/03514-9FAPESP: 07/56614-6CNPq: 470070/2004-8CNPq: 575829/2008-7Web of Scienc

Infestin 1R, an intestinal subtilisin inhibitor from Triatoma infestans able to impair mammalian cell invasion by Trypanosoma cruzi

Infestins are Kazal-type serine protease inhibitors described in the midgut of Triatoma infestans, Chagas disease vector. of all infestins, only infestin 1R (INF1R) does not control host blood coagulation, due to its inhibitory specificity for chymotrypsin-like proteases. We further investigated the effect of INF1R on cell infection by Trypanosoma cruzi. the importance of INF1R reactive site to inhibit T. cruzi cell invasion was confirmed using 1RSFTI, a synthetic cyclic peptide containing the inhibitor reactive site region hybridized to the Sunflower Trypsin Inhibitor-1 (SFTI-1). Our results suggest that INF1R efficiently inhibited parasite cell invasion. for the first time, a serine protease inhibitor, derived from T. infestans, was shown to impair cell invasion by T. cruzi, representing possible new target in parasite cell invasion. (C) 2011 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Bioquim, Escola Paulista Med, São Paulo, BrazilUniv La Habana, Fac Biol, Ctr Estudio Prot, Havana, CubaUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim, Escola Paulista Med, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilFAPESP: 05/03514-9FAPESP: 02/12593-1FAPESP: 09/50434-1CNPq: 470070/2004-8Web of Scienc

Rmcystatin3, a cysteine protease inhibitor from Rhipicephalus microplus hemocytes involved in immune response

The Rhipicephalus microplus tick is responsible for losses in the livestock production estimated in 2 billions USD. Despite its economical importance the knowledge in tick's physiology is sparse. in order to contribute to this scenario we describe the characterization of a cysteine proteinase inhibitor named Rmcystatin-3. Purified recombinant Rmcystatin-3 was able to inhibit cathepsin L (Ki = 2.5 nM), BmCl1 (Ki = 1.8 nM) and cathepsin B (Ki = 136 nM). Western blot and quantitative PCR analysis revealed the presence of Rmcystatin-3 in fat body, salivary gland but mainly in hemocytes. the mRNA levels of Rmcystatin-3 during bacterial challenge are drastically down-regulated. in order to define the Rmcystatin-3 possible role in tick immunity, the cystatin gene was knockdown by RNA interference with and without Escherichia coli infection. Our results showed that the Rmcystatin-3 silenced group was more immune competent to control bacterial infection than the group injected with non-related dsRNA. Taking together, our data strongly suggested an important role of Rmcystatin-3 in tick immunity. (C) 2014 Elsevier B.V. and Societe francaise de biochimie et biologie Moleculaire (SFBBM). All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)INCT-Entomologia Molecular, BrazilUniversidade Federal de São Paulo, Dept Biochem, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniv Fed Rio Grande do Sul, Ctr Biotecnol Estado Rio Grande do Sul, Porto Alegre, RS, BrazilUniversidade Federal de São Paulo, Dept Biochem, Escola Paulista Med, BR-04044020 São Paulo, BrazilFAPESP: 05/03514-9FAPESP: 09/17589-1FAPESP: 12/03657-8Web of Scienc

Production of serine protease inhibitors by mutagenesis and their effects on the mortality of Aedes aegypti L. larvae

BACKGROUND: Dengue, transmitted primarily by the bites of infected Aedes aegypti L., is transmitted to millions of individuals each year in tropical and subtropical areas. Dengue control strategies are primarily based on controlling the vector, using insecticides, but the appearance of resistance poses new challenges. Recently, highly selective protease inhibitors by phage display were obtained for digestive enzymes of the 4th instar larvae (L4) midgut. These mutants were not confirmed as a larvicide due to the low yield of the expression of these inhibitors. In the present study, chimera molecules were constructed based on the mutations at positions P1-P4’ selected previously. The T6, T23 and T149 mutants were mixed with another Kunitz inhibitor, domain 1 of the inhibitor boophilin (D1). METHODS: The chimeras T6/D1, T149/D1 and T23/D1 were expressed at high levels in P. pastoris yeast, purified by ionic exchange chromatography and their homogeneity was analyzed by SDS-PAGE. The chimera inhibitors were assayed against larval trypsin, chymotrypsin and elastase using specific chromogenic substrates. The inhibitors were assayed for their larvicide potential against L4. RESULTS: The chimeras exhibited strong inhibitory activities against the larval digestive enzymes in a dose-dependent manner. T6/D1, T149/D1 and T23/D1 exhibited strong larvicidal activity against L4 of Ae. aegypti with inhibitor concentrations in the μM range. A synergistic increase in mortality was observed when a mixture of the three chimeric inhibitors was tested. CONCLUSIONS: The strategy for constructing the chimeric inhibitors was successful. The chimeras showed strong larvicidal activity against Ae. aegypti. In the future, our findings can be used to design synthetic inhibitors for larvae digestive enzymes as an alternative method to control the dengue vector

Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors

The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech-derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage, A limited combinatorial library of 5.2 x 10(4) mutants was created with a synthetic LDTI gene, using a degenerated oligonucleotide and the pCANTAB 5E phagemid. the mutations were restricted to the P1-P4' positions of the reactive site. Fusion phages and appropriate host strains containing the phagemids were selected after binding to thrombin and DNA sequencing. the variants LDTI-2T (K8R, I9V, S10, K11W, P12A), LDTI-5T (K8R, I9V, S10, K11S, P12L) and LDTI-10T (K8R, I9L, S10, K11D, P12I) were produced with a Saccharomyces cerevisiae expression system. the new inhibitors, LDTI-2T and -5T, prolong the blood clotting time, inhibit thrombin (Ki 302 nM and 28 nM) and trypsin (K-i 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or neutrophil elastase, the variant LDTI-10T binds to thrombin but does not inhibit it, the relevant reactive site sequences of the thrombin inhibiting variants showed a strong preference for arginine in position P1 (K8R) and for valine in P1' (I9V), the data indicate further that LDTI-5T might be a model candidate for generation of active-site directed thrombin inhibitors and that LDTI in general may be useful to generate specific inhibitors suitable for a better understanding of enzyme-inhibitor interactions. (C) 1999 Federation of European Biochemical Societies.UNIFESP, Dept Bioquim, EPM, BR-04044020 São Paulo, BrazilUniv Munich, Klinikum Innenstadt, Chirurg Klin & Poliklin, Klin Chem & Klin Biochem Abt, D-8000 Munich, GermanyUNIFESP, Dept Med, Disciplina Hematol, EPM, BR-04044020 São Paulo, BrazilUNIFESP, Dept Bioquim, EPM, BR-04044020 São Paulo, BrazilUNIFESP, Dept Med, Disciplina Hematol, EPM, BR-04044020 São Paulo, BrazilWeb of Scienc

Protease inhibitors extracted from caesalpinia echinata lam. Affect kinin release during lung inflammation

Inflammation is an essential process in many pulmonary diseases in which kinins are generated by protease action on kininogen, a phenomenon that is blocked by protease inhibitors. We evaluated kinin release in an in vivo lung inflammation model in rats, in the presence or absence of CeKI (C. echinata kallikrein inhibitor), a plasma kallikrein, cathepsin G, and proteinase-3 inhibitor, and rCeEI (recombinant C. echinata elastase inhibitor), which inhibits these proteases and also neutrophil elastase. Wistar rats were intravenously treated with buffer (negative control) or inhibitors and, subsequently, lipopolysaccharide was injected into their lungs. Blood, bronchoalveolar lavage fluid (BALF), and lung tissue were collected. In plasma, kinin release was higher in the LPS-treated animals in comparison to CeKI or rCeEI groups. rCeEI-treated animals presented less kinin than CeKI-treated group. Our data suggest that kinins play a pivotal role in lung inflammation and may be generated by different enzymeshowever, neutrophil elastase seems to be the most important in the lung tissue context. These results open perspectives for a better understanding of biological process where neutrophil enzymes participate and indicate these plant inhibitors and their recombinant correlates for therapeutic trials involving pulmonary diseases.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo [04/11015-0, 07/55496-0, 01/02457-0]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico [304923/2006-0, 304719/2009-9]Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior/Ministerio da Educacao Superior de Cuba (CAPES/MES), Brazil [011/06, 077/09]Department of Biochemistry, Universidade Federal de S˜ao Paulo, Rua Trˆes de Maio, No. 100, 04044-020 S˜ao Paulo, SP, BrazilSchool of Arts, Sciences and Humanities, Universidade de São Paulo, Avenida Arlindo Bettio, No. 1000, 03828-000 São Paulo, SP, BrazilDepartment of Marine Sciences, Universidade Federal de São Paulo, Rua Doutor Carvalho de Mendonça, No. 144, 11070-100 Santos, SP, BrazilJapan Health Care College, Sinei 434-1, Kiyota-ku, Sapporo, JapanDepartment of Marine Sciences, Universidade Federal de São Paulo, Rua Doutor Carvalho de Mendonça, No. 144, 11070-100 Santos, SP, BrazilFAPESP: 04/11015-0FAPESP: 07/55496-0FAPESP: 01/02457-0CNPq: 304923/2006-0CNPq: 304719/2009-9CAPES/MES: 011/06 and 077/09Web of Scienc

Differential transcript profile of inhibitors with potential anti-venom role in the liver of juvenile and adult Bothrops jararaca snake

Background. Snakes belonging to the Bothrops genus are vastly distributed in Central and South America and are responsible for most cases of reported snake bites in Latin America. The clinical manifestations of the envenomation caused by this genus are due to three major activities-proteolytic, hemorrhagic and coagulant-mediated by metalloproteinases, serine proteinases, phospholipases A(2) and other toxic compounds present in snake venom. Interestingly, it was observed that snakes are resistant to the toxic effects of its own and other snake's venoms. This natural immunity may occur due the absence of toxin target or the presence of molecules in the snake plasma able to neutralize such toxins. Methods. In order to identify anti-venom molecules, we construct a cDNA library from the liver of B. jararaca snakes. Moreover, we analyzed the expression profile of four molecules-the already known anti-hemorrhagic factor Bj46a, one gamma-phospholipase A(2) inhibitor, one inter-alpha inhibitor and one C1 plasma protease inhibitor-in the liver of juvenile and adult snakes by qPCR. Results. The results revealed a 30-fold increase of gamma-phospholipase A(2) inhibitor and a minor increase of the inter-alpha inhibitor (5-fold) and of the C1 inhibitor (3-fold) in adults. However, the Bj46a factor seems to be equally transcribed in adults and juveniles. Discussion. The results suggest the up-regulation of different inhibitors observed in the adult snakes might be a physiological adaptation to the recurrent contact with their own and even other snake's venoms throughout its lifespan. This is the first comparative analysis of ontogenetic variation of expression profiles of plasmatic proteins with potential anti-venom activities of the venomous snake B. jararaca. Furthermore, the present data contributes to the understanding of the natural resistance described in these snakes.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Tecnologico (CNPq)INCT - Entomologia MolecularInst Butantan, Lab Herpetol, Sao Paulo, BrazilUniv Sao Paulo, Interunidades Biotecnol, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Bioquim, Sao Paulo, BrazilUniv Fed Rio de Janeiro, Dept Bioquim, Rio De Janeiro, BrazilInst Nacl Ciencia & Tecnol Entomol Mol, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Bioquim, Escola Paulista Med, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Bioquim, Sao Paulo, BrazilFAPESP: 2010/10266-5FAPESP: 2012/03657-8FAPESP: 2013/05357-4FAPESP: 2014/11108-0CNPq: 308780/2013-2Web of Scienc

Protease Inhibitors Extracted from Caesalpinia echinata

Inflammation is an essential process in many pulmonary diseases in which kinins are generated by protease action on kininogen, a phenomenon that is blocked by protease inhibitors. We evaluated kinin release in an in vivo lung inflammation model in rats, in the presence or absence of CeKI (C. echinata kallikrein inhibitor), a plasma kallikrein, cathepsin G, and proteinase-3 inhibitor, and rCeEI (recombinant C. echinata elastase inhibitor), which inhibits these proteases and also neutrophil elastase. Wistar rats were intravenously treated with buffer (negative control) or inhibitors and, subsequently, lipopolysaccharide was injected into their lungs. Blood, bronchoalveolar lavage fluid (BALF), and lung tissue were collected. In plasma, kinin release was higher in the LPS-treated animals in comparison to CeKI or rCeEI groups. rCeEI-treated animals presented less kinin than CeKI-treated group. Our data suggest that kinins play a pivotal role in lung inflammation and may be generated by different enzymes; however, neutrophil elastase seems to be the most important in the lung tissue context. These results open perspectives for a better understanding of biological process where neutrophil enzymes participate and indicate these plant inhibitors and their recombinant correlates for therapeutic trials involving pulmonary diseases

Biochemical Aspects of a Serine Protease from Caesalpinia echinata Lam. (Brazilwood) Seeds: A Potential Tool to Access the Mobilization of Seed Storage Proteins

Several proteins have been isolated from seeds of leguminous, but this is the first report that a protease was obtained from seeds of Caesalpinia echinata Lam., a tree belonging to the Fabaceae family. This enzyme was purified to homogeneity by hydrophobic interaction and anion exchange chromatographies and gel filtration. This 61-kDa serine protease (CeSP) hydrolyses H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide (Km 55.7 μM) in an optimum pH of 7.1, and this activity is effectively retained until 50°C. CeSP remained stable in the presence of kosmotropic anions (PO4 3−, SO4 2−, and CH3COO−) or chaotropic cations (K+ and Na+). It is strongly inhibited by TLCK, a serine protease inhibitor, but not by E-64, EDTA or pepstatin A. The characteristics of the purified enzyme allowed us to classify it as a serine protease. The role of CeSP in the seeds cannot be assigned yet but is possible to infer that it is involved in the mobilization of seed storage proteins