Peptidases are enzymes that hydrolyse peptide bonds. This potentially dangerous activity is regulated by post translational modification and peptidase inhibitors. The best characterized of the peptidase inhibitors are the serpins whilst the aspartic peptidase inhibitors are the least characterized. Aspartic peptidase inhibitors are rare with only nine known sources. However, they are of great interest because they play an important part in several human diseases such as metastasis of breast cancer cells, Candida albicans infections and HIV. The aims of this research project were to investigate the structure of Squash Aspartic peptidase inhibitor (SQAPI), using nuclear magnetic resonance spectroscopy (NMR). This required large amounts of relatively pure and isotopically labeled protein, which was achieved by heterologously expressing His-tagged rSQAPI fusion protein in Escherichia coli using a rich to minimal media transfer method. The fusion protein was purified with a nickel column and the N-terminal extension containing the His6-tag was removed by cleavage of the fusion protein with enterokinase followed by nickel column purification. Preliminary 1 dimensional NMR spectra indicated that SQAPI was folded in solution at pH 3. This was confirmed from the results of a preliminary 15N-edited HSQC. These results combined justified the production of a 15N 13C labeled SQAPI sample for the collection of further NMR spectra. From the spectra produced with double labeled protein the backbone and the side-chain atoms of SQAPI were assigned. The chemical shifts are currently 88.89% complete and have been submitted to the biological magnetic resonance bank (BMRB). A preliminary estimate of the secondary structure of SQAPI has been calculated from the HNHA spectrum suggesting that the SQAPI structure has some similarity to the previously proposed model of the inhibitor’s structure. Furthermore, the region corresponding to the putative binding loop on the model of SQAPI was found to be mobile and deuterium exchange experiments indicate that the SQAPI structure is more globular than open.

Orientadora: Profª Drª Márcia Aparecida Sperança / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2017. / A leishmaniose visceral, causada pelos parasitas das espécies Leishmania infantum e Leishmania donovani, do Velho Mundo, e pela Leishmania infantum chagasi, do Novo Mundo, é uma doença infecciosa e letal se não for tratada. O número de casos desta doença está aumentando no Estado de São Paulo. Uma vez diagnosticada, o tratamento para a leishmaniose visceral tem efeitos colaterais importantes, além da ocorrência de parasitas resistentes aos medicamentos disponíveis. Sabe-se que algumas peptidases desempenham um papel importante na fisiologia e doença causada por parasitas do género Leishmania. A inibição específica destas enzimas pode constituir uma importante estratégia para a produção de potentes agentes antiparasitários. Em bactérias E. coli, inibidores de serino peptidases (ISPs) denominados ecotinas, têm sido descritos, sendo também identificados em espécies de Leishmania. A caracterização funcional da ISP1 e ISP2 de L. major mostrou o seu papel na formação de flagelo de promastigotas e na inibição da elastase de neutrófilos de hospedeiro vertebrado, respectivamente. Portanto, os objetivos deste projeto são a expressão e a purificação das proteínas ISP1 e ISP2 de L. i. chagasi (LcISP1 e LcISP2) recombinantes. A amplificação das sequências que codificam as LcISP1 e LcISP2 foi realizada por PCR a partir do DNA genômico, de L. i. chagasi extraído de uma estirpe de referência cedida pelo Laboratório Nacional de Referência de Espécies de Leishmania da Fiocruz, Rio de Janeiro. Os fragmentos de PCR foram clonados no vetor de expressão bacteriano pET28a e as proteínas recombinantes LcISP2 e LcISP1 foram obtidas na fração solúvel do extrato proteico bacteriano. Após expressão das proteínas recombinantes, elas foram purificadas por cromatografia de afinidade em níquel. / Visceral leishmaniasis, caused by the Old World parasites species Leishmania infantum and Leishmania donovani, and by the New World Leishmania infantum chagasi, is an infectious and lethal disease, if untreated. The number of cases of this disease is increasing in the State of São Paulo. Once diagnosed, treatment for visceral leishmaniasis has important side effects, besides occurrence of parasites resistant to the available drugs. It is known that some peptidases play an important role in physiology and disease caused by parasites of the genus Leishmania. The specific inhibition of these enzymes may constitute an important strategy for producing potent antiparasitic agents. In bacteria E. coli, inhibitors of serine peptidases (ISPs) called ecotins, have been described, being also identified in Leishmania species. Characterization of genes encoding ISP1 and ISP2 of L. major, showed its role in the promastigote flagellum formation and in the inhibition of vertebrate host neutrophil elastase, respectively. Therefore, the aims of this project are to produce L. i. chagasi ISP1 (LcISP1) and ISP2 (LcISP2) recombinant forms and to evaluate the its biochemical activity. Amplification of the LcISP1 and LcISP2 encoding sequences were performed by PCR from L. i. chagasi DNA extracted from a reference strain obtained from the Leishmania collection of the Leishmania National Reference Laboratory of the Instituto Oswaldo Cruz. PCR fragments were cloned into the pET28a bacterial expression vector and the recombinant LcISP1 and LcISP2 proteins were present in soluble bacterial extract fraction. After purification by niquel affinity chromatography, recombinant LcISP1 and LcISP2 proteins e were tested for biochemical activity.

Equistatin from the sea anemone Actinia equina contains a protein domain Eqd2 which inhibits aspartic peptidases and has not been characterized in detail. Recombinant Eqd2 was produced in the yeast expression system, and a protocol for its chromatographic purification was designed. The inhibitory specificity of Eqd2 was determined using a fluorescence inhibition assay, showing that Eqd2 is a highly selective inhibitor of cathepsin D-like and pepsin-like aspartic peptidases of family A1. Furthermore, size exclusion chromatography was used to analyze the Eqd2-peptidase complex and Eqd2 oligomerization in solution. Initial screening of crystallization conditions for Eqd2 was performed towards its structural analysis. This work provides important new information about Eqd2 as a unique type of natural inhibitors of aspartic peptidases. Its interaction mechanism can be exploited in the development of synthetic mimetics for regulation of medically important peptidases. (In Czech) Key words: peptidase inhibitors, proteolytic enzymes, activity and inhibition of enzymes, recombinant expression, protein purification, protein crystallization, equistatin

PDI ("potato cathepsin D inhibitor ") and NID ("novel inhibitor of cathepsin D ") from potato (Solanum tuberosum) belong to the protein family of Kunitz inhibitors (I3 family, Merops database). These 20 kDa isoinhibitors with the typical β-trefoil architecture inhibit aspartic and serine peptidases. In this thesis, the constructs for recombinant expression of PDI and NID in the yeast Pichia pastoris system were prepared and high-producing colonies were selected. Both proteins were identified in the cultivation media by mass spectrometry and N-terminal sequencing. A purification protocol for PDI with three chromatographic steps was designed. Analogous functional properties were demonstrated for the purified recombinant PDI and the native PDI isolated from a natural source. Analysis of the inhibitory specificity showed that PDI is a potent inhibitor of selected aspartic peptidases from the A1 family and serine peptidases from the S1 family, including a relevant enzyme of insect origin. This finding supports the hypothesis that Kunitz inhibitors are involved in plant defense against herbivorous insects. The inhibitors prepared within the project will be used for analysis of the reactive centers against target peptidases by protein crystallography. (In Czech) Key words: proteolytic enzymes, activity...