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

Proteomic study revealed cellular assembly and lipid metabolism dysregulation in sepsis secondary to community-acquired pneumonia

Sepsis is a life-threatening disorder characterized by organ dysfunction and a major cause of mortality worldwide. The major challenge in studying sepsis is its diversity in such factors as age, source of infection and etiology. Recently, genomic and proteomic approaches have improved our understanding of its complex pathogenesis. In the present study, we use quantitative proteomics to evaluate the host proteome response in septic patients secondary to community-acquired pneumonia (CAP). Samples obtained at admission and after 7 days of follow-up were analyzed according to the outcomes of septic patients. The patients' proteome profiles were compared with age-and gender-matched healthy volunteers. Bioinformatic analyses of differentially expressed proteins showed alteration in the cytoskeleton, cellular assembly, movement, lipid metabolism and immune responses in septic patients. Actin and gelsolin changes were assessed in mononuclear cells using immunofluorescence, and a higher expression of gelsolin and depletion of actin were observed in survivor patients. Regarding lipid metabolism, changes in cholesterol, HDL and apolipoproteins were confirmed using enzymatic colorimetric methods in plasma. Transcriptomic studies revealed a massive change in gene expression in sepsis. Our proteomic results stressed important changes in cellular structure and metabolism, which are possible targets for future interventions of sepsis.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, CNPqFAPESPUniv Fed Sao Paulo, Hosp Sao Paulo, Div Infect Dis, Escola Paulista Med, BR-04039032 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biochem, Escola Paulista Med, BR-04023900 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, BR-04023062 Sao Paulo, BrazilUniv Fed Sao Paulo, Intens Care Unit, Hosp Sao Paulo, Escola Paulista Med, BR-04024002 Sao Paulo, BrazilHosp Israelita Albert Einstein, Intens Care Unit, BR-05652900 Sao Paulo, BrazilHosp Sirio Libanes, Intens Care Unit, BR-01409001 Sao Paulo, BrazilUniv Fed Sao Paulo, Hosp Sao Paulo, Div Infect Dis, Escola Paulista Med, BR-04039032 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biochem, Escola Paulista Med, BR-04023900 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, BR-04023062 Sao Paulo, BrazilUniv Fed Sao Paulo, Intens Care Unit, Hosp Sao Paulo, Escola Paulista Med, BR-04024002 Sao Paulo, BrazilFAPESP: 2011/20401-4FAPESP: 2013/15636-8CNPq: 305685/2011-2Web of Scienc

Determinação Da Estrutura Tridimensional De Um Inibidor De Serinoproteases Do Tipo Kazal Do Mosquito Aedes Aegypti, Vetor Da Dengue

The Mosquito Ae. Aegypti Is The Main Vector Of Arboviruses For Humans In Brazil And It Is Responsible For Outbreaks Of Dengue, Chikungunya, Yellow Fever, And Recently It Has Been Involved In The High Number Of Humans Infected With Zika Virus. The Hematophageal Activity Of Ae. Aegypti Allowed It To Develop Several Strategies To Control The Hemostasis Of Vertebrate Host"S. In 2010, Our Group Expressed And Characterized A Serinepeptidase Inhibitor Belonging To Kazal Family Called Aati (Aedes Aegypti Trypsin Inhibitor). The Purified Raati Was Able To Inhibit Trypsin And Affect Coagulation Time. With This Data, The Present Work Had As General Objective The Determination Of Three-Dimensional Structure Of Aati To Understand Its Role In Prolonging Coagulation Time. The Raati Was Purified In Amount And An Inhibitor Preparation Containing 20 Mg/Ml Presenting High Degree Of Homogeneity Was Used In Crystallization Experiments. The Raati Crystallized In 100 Mm Sodium Acetate, Ph 5.5 Containing 25% Peg 3350, 5% Peg 400 AndO Mosquito Ae. Aegypti É O Principal Vetor De Arboviroses Para Humanos, No Brasil E O Responsável Por Surtos De Dengue, Chikungunya, Febre Amarela, E Recentemente Esteve Envolvido No Elevado Numero De Infectados Com Vírus Zika. O Hábito Hematofágico Do Ae. Aegypti Permitiu Que Desenvolve-Se Inúmeras Estratégias De Controle Da Hemostasia Do Seu Hospedeiro Vertebrado. Em 2010, O Nosso Grupo Expressou E Caracterizou Um Inibidor De Serinopeptidase Da Família Kazal Denominado De Aati (Aedes Aegypti Trypsin Inhibitor). O Raati Purificado Foi Capaz De Inibir Tripsina E Afetar O Tempo De Coagulação. De Posse Destes Dados, O Presente Trabalho Teve Como Objetivo Geral Determinar A Estrutura Tridimensional Do Aati Para Entender O Seu Papel No Prolongamento Do Tempo De Coagulação. O Raati Foi Purificado Em Quantidade E Uma Preparação Contendo 20 Mg/Ml Do Inibidor Com Alto Grau De Homogeneidade Foi Utilizado Em Experimentos De Cristalização. O Raati Cristalizou Em 100 Mm Acetato De Sódio, Ph 5,5 Contendo 25% Peg 3350; 5%Dados abertos - Sucupira - Teses e dissertações (2018

Selective inhibitors of digestive enzymes from Aedes aegypti larvae identified by phage display

Dengue is a serious disease transmitted by the mosquito Aedes aegypti during blood meal feeding. It is estimated that the dengue virus is transmitted to millions of individuals each year in tropical and subtropical areas. Dengue control strategies have been based on controlling the vector, Ae. aegypti, using insecticide, but the emergence of resistance poses new challenges. the aim of this study was the identification of specific protease inhibitors of the digestive enzymes from Ae. aegypti larvae, which may serve as a prospective alternative biocontrol method. High affinity protein inhibitors were selected by all of the digestive serine proteases of the 4th instar larval midgut, and the specificity of these inhibitors was characterized. These inhibitors were obtained from a phage library displaying variants of HiTI, a trypsin inhibitor from Haematobia irritans, that are mutated in the reactive loop (P1 P4'). Based on the selected amino acid sequence pattern, seven HiTI inhibitor variants were cloned, expressed and purified. the results indicate that the HiTI variants named T6 (RGGAV) and T128 (WNEGL) were selected by larval trypsin-like (IC50 of 1.1 nM) and chymotrypsin-like enzymes (IC50 of 11.6 nM), respectively. the variants 123 (LLGGL) and 1149 (GGVWR) inhibited both larval chymotrypsin-like (IC50 of 4.2 nM and 29.0 nM, respectively) and elastase-like enzymes (IC50 of 1.2 nM for both). Specific inhibitors were successfully obtained for the digestive enzymes of Ae. aegypti larvae by phage display. Our data also strongly suggest the presence of elastase-like enzymes in Ae. aegypti larvae. the Hill variants T6 and T23 are good candidates for the development as a larvicide to control the vector. (C) 2012 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)INCT - Entomologia MolecularUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, BrazilUniv Estadual Norte Fluminense, Lab Biotecnol, Rio de Janeiro, RJ, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, BrazilFAPESP: 05/03514-9FAPESP: 09/05405-3CNPq: 490574/2006-8Web of Scienc

Expression and functional characterization of boophilin, a thrombin inhibitor from Rhipicephalus (Boophilus) microplus midgut

Rhipicephalus (Boophilus)microplus is an ectoparasite responsible for an important decrease in meat, milk and leather production, caused both by cattle blood loss and by the transmission of anaplasmosis and babesiosis. R. microplus is a rich source of serine protease inhibitors, including the trypsin inhibitors BmTI-A and BmTI-6, the subtilisin inhibitor BmSI, and the recently described thrombin inhibitor, boophilin. Boophilin is a double Kunitz-type thrombin inhibitor, with the unusual ability to form a ternary complex with a second (non-thrombin) serine proteinase molecule. the large-scale expression and purification of boophilin and of its isolated N-terminal (D1) domain in Pichia pastoris, its expression profile, and the effect of RNAi-mediated gene silencing in tick egg production are reported. Full-length boophilin and D1 were expressed at 21 and 37.5 mg/L of culture, respectively. Purified boophilin inhibited trypsin (K-i 0.65 nM), neutrophil elastase (K-i 21 nM) and bovine thrombin (K-i 57 pM), while D1 inhibited trypsin and neutrophil elastase (K-i of 2.0 and 129 nM, respectively), but not thrombin. Boophilin gene silencing using RNAi resulted in 20% reduction in egg weight production, suggesting that the expression of boophilin in this life stage would be important but not vital, probably due to functional overlap with other serine proteinase inhibitors in the midgut of R. microplus. Considering our data, Boophilin could be combining with other antigen in a vaccine production for tick control. (C) 2012 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)INCT-Entomologia MolecularFundacao para a Ciencia e a Tecnologia, PortugalEU-FEDERPOCIUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, SP, BrazilInst Butantan, Lab Fisiopatol, São Paulo, SP, BrazilUniv Porto, IBMC, P-9150180 Oporto, PortugalUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, SP, BrazilFAPESP: 05/03514-9FAPESP: 09/05405-3CNPq: 490574/2006-8Fundacao para a Ciencia e a Tecnologia, Portugal: PTDC/BIA-PRO/70627/2006Fundacao para a Ciencia e a Tecnologia, Portugal: REEQ/564/1310/2005: SFR/BPD/46722/2008Web of Scienc

Validation of a Phage Display Method for Protease Inhibitor Selection Using SFTI and HiTI Synthetic Hybrid Peptides

A recombinant Haematobia irritans irritans trypsin inhibitor (HiTI - Mw 7030 kDa) phagemid library was constructed and displayed functionally on the tip of the filamentous M13 phage. A combinatorial library of 7.2 x 10(6) mutants was created with HiTI mutations restricted to the P1' - P3' and P5' positions of the reactive site. This combinatorial library was selected for trypsin-like Pr2 proteases of Metarhizium anisopliae fungus, and 11 HiTI mutants containing the following substitutions: K17G, S18R, D19G, S21A, among 60 sequenced clones, were obtained. In order to confirm the inhibitory activity of the selected sequences, we transferred the selected sequence to the shortest protease inhibitor, the sunflower trypsin inhibitor (SFTI - Mw 1533 Da), for inhibitory activity analysis. The hybrid peptide containing the mutated sequence (SFTI-Mut, GRCTRGRGLACFPD-NH(2); Ki = 14 mu M) presented an apparent inhibition constant (Ki(app)) for Pr2 proteases similar to 20-fold lower than the control peptide containing the original HiTI sequence (SFTI-HiTI, GRCTRKSDLSCFPD-NH(2); Ki = 259 mu M). In conclusion, the present work enabled the selection of a specific HiTI mutant for Pr2 proteases of M. anisopliae fungus using a HiTI combinatorial library on M13 phage surface. Selection of strong binders by phage display and their validation as inhibitors using synthetic hybrid peptides proved to be a powerful technique to generate specific serine protease inhibitors suitable for studies of drug design and enzyme-inhibitor interaction.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)Univ Fed Sao Paulo UNIFESP, Dept Biochem, BR-04044020 Sao Paulo, BrazilUniv Fed Sao Paulo UNIFESP, Dept Biophys, BR-04044020 Sao Paulo, BrazilUniv Fed Sao Paulo UNIFESP, Dept Biochem, BR-04044020 Sao Paulo, BrazilUniv Fed Sao Paulo UNIFESP, Dept Biophys, BR-04044020 Sao Paulo, BrazilFAPESP: 02/13960-8FAPESP: 05/03514-9FAPESP: 05/03339-2CNPq: 470297/2006-9Web of Scienc

Boophilus microplus cathepsin L-like (BmCL1) cysteine protease: Specificity study using a peptide phage display library

The tick Rhipicephalus (Boophilus) microplus is one of the most important bovine ectoparasites, a disease vector responsible for losses in meat and milk productions. A cysteine protease similar to cathepsin L, named BmCL1, was previously identified in R. microplus gut, suggesting a role of the enzyme in meal digestion. in this work. BmCL1 was successfully expressed in Pichia pastoris system, yielding 54.8 mg/L of culture and its activity was analyzed by synthetic substrates and against a R. microplus cysteine protease inhibitor, Bmcystatin. After rBmCl1 biochemical characterization it was used in a selection of a peptide phage library to determine rBmCL1 substrate preference. Obtained sequenced clones showed that rBmCL1 has preference for Leu or Arg at P(1) position. the preference for Leu at position P(1) and the activation of BmCL1 after a Leu amino acid residue suggest possible self activation. (C) 2011 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)INCT-Entomologia MolecularUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilUniv Fed Rio Grande do Sul, Ctr Biotecnol Estado Rio Grande Sul, Porto Alegre, RS, 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: 05/03339-2FAPESP: 09/50434-1CNPq: 470297/2006-9Web of Scienc