Orientador: Sergio Marangoni / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-24T16:20:58Z (GMT). No. of bitstreams: 1 Pando_SilvanaCristina_M.pdf: 4387966 bytes, checksum: 19da59ac17254dba0a8949838880eefc (MD5) Previous issue date: 1999 / Resumo: No presente trabalho foi investigado um inibidor de proteinases em sementes da espécie Delonix regia Leguminosae-Caesalpinioideae). As sementes das leguminosas contêm uma alta concentração de proteína em relação aos outros componentes da planta. Dentre essas proteínas, os inibidores de proteinases, particularmente inibidores de enzimas da família das serinoproteinases (que inclui tripsina, quimotripsina e enzimas da coagulação sanguínea), exercem uma importante função na fisiologia da planta. Os inibidores de proteinases têm sido estudados há mais de 50 anos. Eles têm mostrado ser muito importantes no tratamento de disfunções metabólicas associadas às enzimas proteolíticas, tais como em casos de pancreatites, efizemas, alergias, inflamação, hipertensão e câncer. O inibidor de Delonix regia (DrTI) foi isolado em solução salina 10% (p/v), submetido a uma precipitação por acetona 80% (v/v) e purificado através de cromatografias de troca iônica (Sephadex A-50 e DEAE 5PW) e de exclusão molecular (Sephadex G-75). o DrTI inibiu tripsina bovina e porcina e a calicreína plasmática humana. Os valores de Ki encontrados foram 2, 19x1 0-8M, 4,53x10-8M e 5,25x 10-9 M, respectivamente. A pureza do inibidor foi verificada por eletroforese em gel de poliacrilamida (10-20%), na presença de 5DS. O inibidor apresentou uma única banda de proteína em condições nativas e reduzidas (0,1 M DTT). Estes resultados indicam que o DrTI é constituído por uma única cadeia polipeptídica. A massa molecular aparente do inibidor foi de 22 kDa, um resultado similar aos inibidores de proteinase do tipo Kunitz. O DrTI foi submetido a estudos cristalográficos e seu modelo permitiu comparação estrutural com o inibidor de tripsina do tipo Kunitz isolado de sementes de Erythrina caffra / Abstract: In the present work, a proteinase inhibitor in seeds of Delonix regia (LeguminosaeCaesalpinioideae) specie, was investigated. The Leguminosae seeds contain a high concentration of protein in relation to the other components of the plant. Among these proteins, the proteinase inhibitors, particularly enzymes inhibitors of serinoproteinases family (that include trypsin, chymotrypsin and blood coagulating enzymes), play an important role in plant physiology. Plant inhibitors have been investigated for more than fifty years ago. They have been shown to be very important in the treatment of metabolic disfunctions associated to proteolytic enzymes, such as in cases of pancreatits, emphyzema, allergy, inflamation and cancers. The Delonix regia inhibitor (DrTI) was isolated in saline solution 10% (p/v), after acetonic precipitation 80% (v/v), and purified through ion exchange chromatography (Sephadex A-50 e DEAE 5PW) and molecular exclusion (Sephadex G-75). DrTI inhibited bovine and porcine trypsin and human plasma kallikrein. The values of Ki found were 2,19 x 10-8M, 4,53 x 10-8M and 5,25 x 10-9M, respectively. Purity of inhibitor was detected by poliacrylamide gel electrophoresis (10-20%) in the presence of SDS. The inhibitor displayed a single protein band, in native and reduced conditions (0,1 M DTT). These results indicate that DrTI is constituted by a single polipeptide chain.The apparent molecular mass of inhibitor was of 22 kDa, a result similar to the proteinase inhibitors of the Kunitz-type. DrTI was submitted to crystallographic studies and its model allowed structural comparison to the trypsin inhibitor of the Kunitz-type isolated from Erythrina caffra seeds / Mestrado / Bioquimica / Mestre em Ciências Biológicas

A aprotinina, um inibidor de serinoproteinase ácido resistente de massa molar de 7 kDa, é utilizada como insumo ou medicamento. O objetivo principal deste trabalho foi purificar a aprotinina a partir de pulmão suíno. Três procedimentos foram utilizados. O primeiro procedimento utilizou a coluna de tripsina-agarose, o segundo procedimento utilizou a filtração tangencial e coluna de tripsina-Sepharose. O terceiro procedimento utilizou três cromatografias: filtração em gel, troca-iônica e afinidade (tripsina-agarose). A aprotinina suína foi purificada de pulmão utilizando o terceiro procedimento. A seqüência parcial do gene da aprotinina suína apresentou 74% de identidade com a seqüência do gene da aprotinina bovina. Outros dois inibidores de serinoproteinases ácido resistentes foram purificados, são eles: o fragmento ativo do segundo domínio do inibidor de leucoprotease secretada (SLPI), e um segundo inibidor de alta massa molecular, provavelmente bikunina. O protocolo de purificação utilizado neste trabalho recuperou 85mg de aprotinina suína por kg de pulmão. / Aprotinin, an acid stable serine proteinase inhibitor with a molecular mass of 7 kDa, is used as a reagent or drug. The purification of the aprotinin from porcine lungs was the main objective of this work. Three procedures were used. The first one utilized the trypsin-agarose column. The tangential ultra filtration and trypsin-Sepharose column were used in the second procedure. And finally, the gel filtration, ion-exchange and affinity chromatography were employed in the third procedure. The porcine lung aprotinin was purified using the third procedure. The partial sequence of the aprotinin gene was obtained and showed 74% of the identity with the aprotinin bovine gene sequence. Another two acid stable serine proteinase inhibitors were purified: the active fragment of the secretory leukoprotease inhibitor second domain, and one high molecular mass inhibitor, probably bikunina. The purification protocol used in this work recovered 85mg of the porcine aprotinin from kg of lung.

Para conseguirem se alimentar com sucesso, os mosquitos hematófagos possuem componentes em sua saliva capazes de regular a hemostasia e modular a imunidade dos hospedeiros. Entretanto, a avaliação das atividades biológicas dessas moléculas no hospedeiro ainda carece de estudos mais aprofundados. No presente projeto, propomos caracterizar as atividades biológicas do produto do transcrito AET-7393, uma serpina presente nas glândulas salivares de fêmeas do mosquito Aedes aegypti. Nossos dados mostram que a serpina AET-7393 recombinante provoca um aumento no sangramento quando inoculada em camundongos, mas aparentemente esse efeito não está ligado à interferência com a cascata de coagulação. Mostramos ainda que a AET-7393 é capaz de inibir a proteinase 3 e aumentar a produção de IL-1b. Por fim, observamos a ausência de capacidade moduladora sobre a ativação de macrófagos ou sobre a inflamação, e que presença de anticorpos específicos contra a serpina no hospedeiro não interfere no ciclo de vida do mosquito. / In order to successfully feed, hematophagous mosquitoes possess salivary components capable of regulating hemostasis and modulate the host immunity. However, the evaluation of the biological activities of the salivary molecules in the host still needs further investigation. In this study, we intend to characterize the biological activities of the AET-7393, a serpin that is present in the saliva of the females Aedes aegypti mosquitoes. Our data show that the recombinant AET-7393 serpin increases bleeding when inoculated in mice, but apparently this effect is not due to its interference on the coagulation cascade. In addition, AET-7393 is able to inhibit proteinase 3 and enhance the production of IL-1b. Finally, we observed the absence of modulatory effect on macrophage activation or inflammation, and that the presence of host anti-AET-7393 antibodies does not interfere in the life cycle of the mosquitoes.

Thesis (M.Sc. (Biochemistry)) -- University of Limpopo, 2011 / Soybean is one of the most cultivated legume plants in developing countries. Nodule senescence is a major limitation in producing high yields of soybean as it coincides with the pod filling stage. Delaying nodule senescence could be a way of increasing the yield of soybean therefore determination of the role of cysteine protease in soybean is of vital importance. In this study, soybean plants were grown under controlled temperature and light conditions. Leaves and root crown nodules were collected at 4, 6, 10, 12 and 16 weeks of age. In a comparative 1-dimensional SDS-PAGE analysis of soybean nodule proteomes as the plant matured, it showed differences in proteins expressed as shown by different banding patterns with less variation between the younger soybean nodule extracts (4, 6 and 10 weeks old) as compared to the older ones (12 and 16 weeks old). As determined by azocasein assay and protease zymography, the protease activity of the nodule extracts generally decreased with an increase in the age of the nodules whereas that of the leaves increased as the plants grew older. Cysteine proteases in the soybean nodule extracts readily cleaved the Z-Arg-Arg-AMC substrate with the highest activity shown in the younger nodules as compared to the older ones. In the leaf extracts, cysteine protease activity increased with age of the leaves. DCG-04, a biotinylated irreversible inhibitor, proved to be an effective label in profiling of activity of cysteine proteases in 1-dimensional and 2-dimensional systems. The labelling was inhibited specifically by cysteine protease inhibitor, E-64. In root nodules, the DCG-04 probing demonstrated that the expression of cysteine proteases is higher in early stages of development of the soybean nodules as compared to the later stages whereas in the leaves, there is higher expression of cysteine proteases in the old leaves (16 weeks). Using 2-dimensional polyacrylamide gel electrophoresis, five cysteine protease isoforms were visualised with the size ranging from approximately 25 to 30 kDa and a pI range of 4-6. In older nodules (12 and 16 weeks old) the higher pI isoforms are down-regulated with the 26 kDa and pI 4.5 protease being the predominant isoform. Affinity precipitation of the cysteine proteases yielded a strong band with the size of about 26 kDa. All assays used show that while in leaves, the expected trend of high expression of cysteine proteases in senescing leaves is observed, in soybean nodules the expression of cysteine proteases decreases with senescence. There is, therefore, no correlation between senescence and cysteine proteases in nodules. The highly expressed cysteine protease in young nodules could play a developmental or regulatory role during the early stages of development.

Cysteine proteases are important drug targets due to their involvement in many biological processes such as protein turnover, digestion, blood coagulation, apoptosis, cell differentiation, cell signaling, and the immune response. In this thesis, we have reported the design, synthesis and evaluation of clan CA and clan CD cysteine protease inhibitors. Aza-peptidyl Michael acceptor and epoxide inhibitors for asparaginyl endopeptidases (legumains) from the bloodfluke, Schistosoma mansoni (SmAE) and the hard tick, Ixodes ricinus (IrAE) were designed and synthesized. SARs were similar, but with some notable exceptions. Both enzymes prefer disubstituted amides to monosubstituted amides in the P1' position and potency increased as we increased the hydrophobicity of the inhibitor in this position. Extending the inhibitor to P5 resulted in increased inhibitory potency, especially against IrAE, and both enzymes prefer small over large hydrophobic residues in the P2 position. Aza-peptide Michael acceptor inhibitors are more potent than aza-peptide epoxide inhibitors and, for some of these compounds, second order inhibition rate constants are the fastest yet discovered. We have also synthesized aza-peptidyl Michael acceptor and epoxide inhibitors for the parasitic cysteine proteases; cruzain, rhodesain. We have found that monosubstituted amides were favored over disubstituted amides indicating the involvement of the amide hydrogen in a H-bond network. We have shown that aza-peptide epoxides were as potent as Michael acceptors and we have obtained compounds with IC50 values as low as 20 nM. We have worked on the synthesis of heterocyclic peptidyl α-ketoamides, peptidyl ketones and aza-peptidyl ketones as calpain inhibitors. We have synthesized peptidyl α-ketoamides with nucleotide bases in the primed region to create compounds that can cross the blood-brain barrier. We have improved the potency by introducing a hydrophobic group on the adenine ring. We have obtained compounds with Ki values in the nanomolar range. We have designed peptidyl aminoketones as a new class of inhibitors for calpain. Peptidyl aminoketones were less potent than peptidyl α-ketoamides but still reasonable inhibitors of calpain that have the potential to cross the BBB.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 143-164). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 143-164). Also available on the Internet.

Thesis (Ph.D.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Chemistry. Title from pdf file main screen (viewed on November 8, 2009). Includes bibliographical references.

Malaria is an infectious disease caused by parasites of the Plasmodium genus with mortality rates of more than a million annually, hence a major global public health concern. Plasmodium falciparum (P. falciparum) accounts for over 90% of malaria incidence. Increased resistance to antimalarial drugs by the Plasmodium parasite, coupled with the lack of an effective malaria vaccine necessitates the urgent need for new research avenues to develop novel and more potent antimalarial drugs. This study focused on falcipains, a group of P. falciparum cysteine proteases that belong to the clan CA and papain family C1, that have emerged as potential drug targets due to their involvement in a range of crucial functions in the P. falciparum life cycle. Recently, falcipain-2 has been validated as a drug target but little is known of its Plasmodium orthologs. Currently, there are several falcipain inhibitors that have been identified, most of which are peptide based but none has proceeded to drug development due to associated poor pharmacological profiles and susceptibility to degradation by host cysteine proteases. Non-peptides inhibitors have been shown to be more stable in vivo but limited information exists. In vivo studies on falcipain-2 and falcipain-3 inhibitors have also been complicated by varying outcomes, thus a good understanding of the structural variations of falcipain Plasmodium orthologs at the active site could go a long way to ease in vivo results interpretation and effective inhibitor design. In this study, we use bioinformatics approaches to perform comparative sequence and structural analysis and molecular docking to characterize protein-inhibitor interactions of falcipain homologs at the active site. Known FP-2 and FP-3 small molecule nonpeptide inhibitors were used to identify residue variations and their effect on inhibitor binding. This was done with the aim of screening a collection of selected non-peptide compounds of South African natural origin to identify possible new inhibitor leads. Natural compounds with high binding affinities across all Plasmodium orthologs were identified. These compounds were then used to search the ZINC database for similar compounds which could have better binding affinities across all selected falcipain homologs. Compounds with high binding affinities across all Plasmodium orthologs were found.

Para conseguirem se alimentar com sucesso, os mosquitos hematófagos possuem componentes em sua saliva capazes de regular a hemostasia e modular a imunidade dos hospedeiros. Entretanto, a avaliação das atividades biológicas dessas moléculas no hospedeiro ainda carece de estudos mais aprofundados. No presente projeto, propomos caracterizar as atividades biológicas do produto do transcrito AET-7393, uma serpina presente nas glândulas salivares de fêmeas do mosquito Aedes aegypti. Nossos dados mostram que a serpina AET-7393 recombinante provoca um aumento no sangramento quando inoculada em camundongos, mas aparentemente esse efeito não está ligado à interferência com a cascata de coagulação. Mostramos ainda que a AET-7393 é capaz de inibir a proteinase 3 e aumentar a produção de IL-1b. Por fim, observamos a ausência de capacidade moduladora sobre a ativação de macrófagos ou sobre a inflamação, e que presença de anticorpos específicos contra a serpina no hospedeiro não interfere no ciclo de vida do mosquito. / In order to successfully feed, hematophagous mosquitoes possess salivary components capable of regulating hemostasis and modulate the host immunity. However, the evaluation of the biological activities of the salivary molecules in the host still needs further investigation. In this study, we intend to characterize the biological activities of the AET-7393, a serpin that is present in the saliva of the females Aedes aegypti mosquitoes. Our data show that the recombinant AET-7393 serpin increases bleeding when inoculated in mice, but apparently this effect is not due to its interference on the coagulation cascade. In addition, AET-7393 is able to inhibit proteinase 3 and enhance the production of IL-1b. Finally, we observed the absence of modulatory effect on macrophage activation or inflammation, and that the presence of host anti-AET-7393 antibodies does not interfere in the life cycle of the mosquitoes.