Resumo: Metaloproteinases presentes no veneno de serpentes são toxinas hemostaticamente ativas que interferem em diferentes níveis na cascata de coagulação e têm sido exploradas como ferramenta bioquímica nas pesquisas de coagulação, diagnósticos, modelos de inibidores de metaloproteinases e compreensão do mecanismo de ação de venenos. Estudos estruturais complementados por ensaios de docking molecular têm ajudado muito na compreensão de suas especificidades e mecanismo de ação. Assim, neste trabalho a metaloproteinase não-hemorrágica fibrinogenolítica da classe PI, isolada do veneno de Bothrops moojeni (BmooMP -I) foi cristalizada e sua estrutura resolvida a 1,76 Å de resolução. O íon zinco catalítico possui uma coordenação octaédrica formada por três histidinas canônicas (His 140 , His 144 e His 150 ) e por três moléculas de solvente. Uma comparação seqüencial e estudos estruturais indicaram que os motivos que compreendem os resíduos 153-164 e 167-176 possuem uma característica saliente, que diferencia as outras SVMPs hemorrágicas das não-hemorrágicas de classe PI, podendo estes motivos estarem diretamente envolvidos no desenvolvimento da atividade hemorrágica. Além do projeto principal, foram realizados estudos com a proteína endo-1,4-β-glicanase de Bacillus subtilis (Cel5A), que hidrolisa a ligação glicosídica β(1-4) do polímero de celulose. Esse polímero tem recebido grande atenção do meio acadêmico e industrial devido ao seu potencial na produção de açúcares solúveis, produtos químicos e biocombustíveis. Porém para o aproveitamento da biomassa celulósica é necessário a ação sinérgica do coquetel enzimático formado por endo-1,4-β-glicanases, celobiose-hidrolases ou exo-1,4-β-glicanases e β-glicosidases. Assim, o gene Cel5A constituído pelo domínio catalítico e o módulo de ligação à carboidratos (CBM) foi clonado... (resumo completo, clicar acesso eletrônico abaixo) / Abstract: Snake venom metalloproteinases are hemostatically active toxins that interfere in different levels on the blood coagulation cascade. Therefore, they have been extensively investigated as biochemical tools for coagulation studies and diagnostics, and as model for design of metalloproteinases inhibitors and understanding of venom action mechanism. Structural studies complemented by molecular docking experiments have been instrumental to shed light on the the stereo specificity and action of enzymes. In this study, a fibrin(ogen)olytic non-hemorragic PI class metalloproteinase isolated from Bothrops moojeni (BmooMPα-I) was crystallized and its structure determined at 1,76 Å resolution. The catalytic zinc ion displays an octahedral coordination formed by three canonical histidines (His 140 , His 144 e His 150 ) and three solvent molecules. Comparative sequence and structural studies indicate that the motif comprising amino acid segments 153-164 e 167-176 is a salient feature that differentiates non- and hemorrhagic PI class SVMPs and it might be directly involved in the development of the hemorrhagic activity. In parallel to the main project, a biophysical study was carried out on the thermophilic endo-1,4-β-glucanase from Bacillus subtilis. Endo-cellulases are responsible for the cleavage of β(1-4) glycosidic linkages from cellulose. This polymer has been receiving great attention from both industrial and academic community due to its applications in food, chemical and biofuels industries. However, for its use is needed to reduce the cellulose into fermentable sugars and this process involves the synergistic action of endo-1,4-β-glucanases, celobiose-hydrolases or exo-β-1,4-glucanases and β-glucosidases. In this project, the gene of the Cel5A consisting of the parental catalytic domain and its carbohydrate-binding module was cloned in pET28a vector and expressed in BL21(DE3)... (Abstract complete click electronic access below) / Orientador: Mário Tyago Murakami / Coorientador: João Ruggiero Neto / Banca: Fernanda Canduri / Banca: Marinonio Lopes Cornélio / Mestre

Made available in DSpace on 2014-06-11T19:22:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-04-08Bitstream added on 2014-06-13T20:29:19Z : No. of bitstreams: 1 akao_pk_me_sjrp.pdf: 3271201 bytes, checksum: 442f358fe13f76b52392394d1b1519df (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Metaloproteinases presentes no veneno de serpentes são toxinas hemostaticamente ativas que interferem em diferentes níveis na cascata de coagulação e têm sido exploradas como ferramenta bioquímica nas pesquisas de coagulação, diagnósticos, modelos de inibidores de metaloproteinases e compreensão do mecanismo de ação de venenos. Estudos estruturais complementados por ensaios de docking molecular têm ajudado muito na compreensão de suas especificidades e mecanismo de ação. Assim, neste trabalho a metaloproteinase não-hemorrágica fibrinogenolítica da classe PI, isolada do veneno de Bothrops moojeni (BmooMP -I) foi cristalizada e sua estrutura resolvida a 1,76 Å de resolução. O íon zinco catalítico possui uma coordenação octaédrica formada por três histidinas canônicas (His 140 , His 144 e His 150 ) e por três moléculas de solvente. Uma comparação seqüencial e estudos estruturais indicaram que os motivos que compreendem os resíduos 153-164 e 167-176 possuem uma característica saliente, que diferencia as outras SVMPs hemorrágicas das não-hemorrágicas de classe PI, podendo estes motivos estarem diretamente envolvidos no desenvolvimento da atividade hemorrágica. Além do projeto principal, foram realizados estudos com a proteína endo-1,4-β-glicanase de Bacillus subtilis (Cel5A), que hidrolisa a ligação glicosídica β(1-4) do polímero de celulose. Esse polímero tem recebido grande atenção do meio acadêmico e industrial devido ao seu potencial na produção de açúcares solúveis, produtos químicos e biocombustíveis. Porém para o aproveitamento da biomassa celulósica é necessário a ação sinérgica do coquetel enzimático formado por endo-1,4-β-glicanases, celobiose-hidrolases ou exo-1,4-β-glicanases e β-glicosidases. Assim, o gene Cel5A constituído pelo domínio catalítico e o módulo de ligação à carboidratos (CBM) foi clonado... / Snake venom metalloproteinases are hemostatically active toxins that interfere in different levels on the blood coagulation cascade. Therefore, they have been extensively investigated as biochemical tools for coagulation studies and diagnostics, and as model for design of metalloproteinases inhibitors and understanding of venom action mechanism. Structural studies complemented by molecular docking experiments have been instrumental to shed light on the the stereo specificity and action of enzymes. In this study, a fibrin(ogen)olytic non-hemorragic PI class metalloproteinase isolated from Bothrops moojeni (BmooMPα-I) was crystallized and its structure determined at 1,76 Å resolution. The catalytic zinc ion displays an octahedral coordination formed by three canonical histidines (His 140 , His 144 e His 150 ) and three solvent molecules. Comparative sequence and structural studies indicate that the motif comprising amino acid segments 153-164 e 167-176 is a salient feature that differentiates non- and hemorrhagic PI class SVMPs and it might be directly involved in the development of the hemorrhagic activity. In parallel to the main project, a biophysical study was carried out on the thermophilic endo-1,4-β-glucanase from Bacillus subtilis. Endo-cellulases are responsible for the cleavage of β(1-4) glycosidic linkages from cellulose. This polymer has been receiving great attention from both industrial and academic community due to its applications in food, chemical and biofuels industries. However, for its use is needed to reduce the cellulose into fermentable sugars and this process involves the synergistic action of endo-1,4-β-glucanases, celobiose-hydrolases or exo-β-1,4-glucanases and β-glucosidases. In this project, the gene of the Cel5A consisting of the parental catalytic domain and its carbohydrate-binding module was cloned in pET28a vector and expressed in BL21(DE3)... (Abstract complete click electronic access below)

Orientador: Raghuvir Krishnaswamy Arni / Banca: Adélia Cristina Oliveira Cintra / Banca: Patrick Jack Spencer / Abstract: Toxins isolated from vemos have been used as molecular tools to understand many physiological processes. The enzymes isolated from the venoms of Crotalus and Bothrops species interfere with the control and balance of the hemostatic system (PEREZ et al., 1996) and thus, the determination of their structures is potentially very important. These enzymes are serine proteinases that are similar to tyrpsin in their specificity but are generally referred to as thrombin-like enzymes due to their ability to cleave fibrinogen. The principal aim of this project was to isolate and characterize snake venom poteins that inetefere with the control and regulation of the hemostatic system in quantities and purity required for structural studies. Gel filtration, ion-exchange and HPLC chromatographic techniques were used to isolate convulxin, crotoxin, giroxin and crotamine, the principle components from the venoms of Crotalus durissus collineatus and Crotalus durissus terrificus and the serine and metalo proteinases from the venom of Bothrops jararaca. The purity of the samples was evaluated by SDS-PAGE and the specific activity of the samples was determined. Crystallization experiments were then carried out. / Mestre

Made available in DSpace on 2014-06-11T19:22:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-12-08Bitstream added on 2014-06-13T20:10:06Z : No. of bitstreams: 1 oliveira_dgl_me_sjrp.pdf: 2024541 bytes, checksum: 9e617f882421a4dd2f2cc715da6fb79f (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Toxins isolated from vemos have been used as molecular tools to understand many physiological processes. The enzymes isolated from the venoms of Crotalus and Bothrops species interfere with the control and balance of the hemostatic system (PEREZ et al., 1996) and thus, the determination of their structures is potentially very important. These enzymes are serine proteinases that are similar to tyrpsin in their specificity but are generally referred to as thrombin-like enzymes due to their ability to cleave fibrinogen. The principal aim of this project was to isolate and characterize snake venom poteins that inetefere with the control and regulation of the hemostatic system in quantities and purity required for structural studies. Gel filtration, ion-exchange and HPLC chromatographic techniques were used to isolate convulxin, crotoxin, giroxin and crotamine, the principle components from the venoms of Crotalus durissus collineatus and Crotalus durissus terrificus and the serine and metalo proteinases from the venom of Bothrops jararaca. The purity of the samples was evaluated by SDS-PAGE and the specific activity of the samples was determined. Crystallization experiments were then carried out.