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

The full-length cDNA of anticoagulant protein infestin revealed a novel releasable Kazal domain, a neutrophil elastase inhibitor lacking anticoagulant activity

Infestins are Kazal-type serine proteinase inhibitors found in the midgut of the Chagas' disease vector, Triatoma infestans. in previous studies, we characterized two double-headed infestins with potent anticoagulant activity; infestin 1-2, which inhibits thrombin and infestin 3-4, a factor XIIa inhibitor. in the present work, we have cloned the full-length cDNA of infestins' precursor. the translated cDNA predicted a polypeptide containing a signal peptide and seven Kazal-type domains, four domains from infestin 1-2 and infestin 3-4, and three new domains. Northern blot analysis confirmed that infestins are synthesized in a single transcript (similar to 1800 bp) in the insect midgut, but not in salivary glands. Based on the cDNA sequence, the three new Kazal domains were named infestin 1R, 2R and 3R. Infestin 2R-3R has 77% amino acid sequence identity to infestin 1-2 and the same basic amino acid residue at PI position in the inhibitory reactive site suggesting that these two proteins have a similar inhibitory specificity. in contrast, infestin IR has two different characteristics when compared to the other infestins: i) a hydrophobic amino acid residue at PI position in the inhibitory reactive site and ii) a prediction to be processed as a single Kazal domain, These two characteristics were experimentally demonstrated by the purification of native infestin I R from T infestans midgut. Native infestin I R was shown to be processed as a single Kazal domain by mass spectrometry and it was able to inhibit neutrophil elastase, subtilisin A and chymotrypsin. To further characterize infestin IR inhibitory activity, it was expressed as a recombinant protein in bacteria. Recombinant infestin IR inhibited neutrophil elastase with the same K-i of the native inhibitor. Moreover, it inhibited subtilisin A, chymotrypsin and proteinase K but did inhibit neither thrombin nor coagulation assays. in conclusion, unlike the other described infestins, infestin IR did not present anticoagulant activity and is processed as a single Kazal domain with inhibitory specificity towards proteases that hydrolyze peptide bonds after hydrophobic amino acid residues. (c) 2006 Elsevier SAS. All rights reserved.Universidade Federal de São Paulo, Dept Biochem, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, Escola Paulista Med, BR-04044020 São Paulo, BrazilWeb 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

NEK1 kinase domain structure and its dynamic protein interactome after exposure to Cisplatin

NEK family kinases are serine/threonine kinases that have been functionally implicated in the regulation of the disjunction of the centrosome, the assembly of the mitotic spindle, the function of the primary cilium and the DNA damage response. NEK1 shows pleiotropic functions and has been found to be mutated in cancer cells, ciliopathies such as the polycystic kidney disease, as well as in the genetic diseases short-rib thoracic dysplasia, Mohr-syndrome and amyotrophic lateral sclerosis. NEK1 is essential for the ionizing radiation DNA damage response and priming of the ATR kinase and of Rad54 through phosphorylation. Here we report on the structure of the kinase domain of human NEK1 in its apo- and ATP-mimetic inhibitor bound forms. The inhibitor bound structure may allow the design of NEK specific chemo-sensitizing agents to act in conjunction with chemo- or radiation therapy of cancer cells. Furthermore, we characterized the dynamic protein interactome of NEK1 after DNA damage challenge with cisplatin. Our data suggest that NEK1 and its interaction partners trigger the DNA damage pathways responsible for correcting DNA crosslinks