Orientadores: Sergio Marangoni, Luis Alberto Ponce Soto / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T16:00:07Z (GMT). No. of bitstreams: 1 Rojas-Hualpa_JoseMiguel_M.pdf: 2266212 bytes, checksum: ad52a054d91af49b26ef11cf744381fe (MD5) Previous issue date: 2014 / Resumo: O resumo, na íntegra poderá ser visualizado no texto completo da tese digital / Abstract: The complete abstract is available with the full electronic document / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientador: Marcos Hikari Toyama / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T14:30:21Z (GMT). No. of bitstreams: 1 Soares_VeronicaCristinaGomes_D.pdf: 49857534 bytes, checksum: b3727eb6c966531052574dc180e90fd1 (MD5) Previous issue date: 2014 / Resumo: Mesmo após décadas do descobrimento dos anti-inflamatórios inibidores de fosfolipase continua a busca por novas moléculas que sejam capazes de apresentar esse efeito terapêutico sem a indução de efeitos colaterais. O objetivo deste estudo foi obter extrato polar de partes aéreas de Tithonia diversifolia, viabilizando um padrão de qualidade para esse extrato a partir da identificação de seus principais constituintes e após essa determinação, avaliar o seu efeito sobre a atividade de frações de fosfolipase A2 básicas secretórias (sPLA2) obtidas de Bothrops jararacussu (Bj) e Crotalus durissus ssp (Cd). Os extratos foram obtidos por infusão e decocção e após a identificação dos constituintes por cromatografia e LC/MS-MS, determinou-se que o extrato de melhor rendimento foi obtido por decocção e que seus principais constituintes são derivados do ácido cinâmico: cafeoil-glicosídico, tagitinina C e ácido quínico. A técnica de molecular imprint (MP) permitiu uma separação dos constituintes sendo uma metodologia prática. O extrato polar apresentou atividade anti-agregante plaquetária na concentração de 0,6 a 20µg/mL, frente ao estímulo de indução por trombina, o que representa que o chá não é tão inerte e que pode promover complicações em indivíduos com distúrbios de coagulação. A purificação de Bj e Cd foi realizada por técnicas cromatográficas, com rendimento de aproximadamente 30% (p<0,05) de sPLA2. Ensaio enzimático in vitro, que utilizou substrato cromogênico sintético de PLA2 o 4-nitro-3-octanoiloxi-benzóico (NOBA) identificou que o extrato polar foi capaz de reduzir em 60% (p<0,05) a atividade enzimática de sPLA2 de Bj, independente do tipo de tratamento aplicado. A redução da atividade de sPLA2 de Cd foi reduzida em 30% (p<0,05) quando a mesma foi incubada por 30 min em presença do extrato. As modificações dos efeitos patológicos causados por sPLA2s foram avaliadas por ensaios in vivo. A ação edematogênica de sPLA2 de Bj foi reduzida em 50% (p<0,05), quando esta estava em presença do extrato, independe de prévia incubação, já a mesma ação para sPLA2 de Cd foi reduzida drasticamente pelo extrato, no entanto, após 60 min da administração do agente indutor de inflamação, a capacidade miotóxica com consequente liberação de creatina-quinase (CK) foi modulada pelo extrato de forma distinta dependendo da fonte de sPLA2. Para sPLA2 de Bj o extrato promoveu uma atividade protetora de liberação de CK, pois reduziu a liberação da enzima quando foi aplicado intraperitoneal 30 min antes do agente agressor, no entanto, a sPLA2 de Cd apresentou capacidade reduzida de liberação de CK quando foi incubada com o extrato por 30min antes da aplicação do mesmo no músculo do cobaio. Diante da ação do extrato sobre a inflamação induzida por sPLA2 buscou-se, através de técnica da reação da polimerase em cadeia em tempo real (PCR em tempo real), determinar a influência do extrato na expressão de genes envolvidos na inflamação. Determinou-se que o gene Nf-Kb, embora responda a presença do extrato, é o que mais tardiamente é ativado (ou expresso), possivelmente por ser nuclear. Através desses ensaios foi possível determinar a ação desses extratos sobre a resposta inflamatória aguda induzida pela ação de sPLA2s de venenos de serpentes / Abstract: Even after decades of the discovery of the anti-inflammatory inhibitors of phospholipase, there is a continuous search for new molecules which are able to provide this therapeutic effect without inducing side effects. The aim of this study was to obtain polar extract of the aerial parts of Tithonia diversifolia, enabling a quality standard for this extract from the identification of its main constituents and after this determination, to evaluate its effect on the activity of fractions A2 phospholipase basic secretory (sPLA2) obtained from Bothrops jararacussu (Bj) and Crotalus durissus ssp (Cd). The extracts were obtained by decoction and infusion and after the identification of the chromatography and LC / MS-MS. It was determined that the best yield of extract was obtained by decoction and its main constituents are cinnamic acid derivatives: caffeoyl - glycosidic, tagitinina C and quinic acid. Molecular imprint technique (MP) enabled separation of the constituents being a practical methodology. The polar extract showed anti-platelet activity at a concentration of 0.6 to 20 ?g/mL, opposite the stimulus induced by thrombin, which indicates that tea is not as inert and may promote complications in patients with coagulation disorders. Purification of Bj and Cd was performed by chromatographic techniques, with a yield of approximately 30% (p<0,05) of sPLA2. In vitro enzyme assay, which used synthetic chromogenic substrate of sPLA2 4-nitro-3-octanoyloxy benzoic acid (NOBA), it was identified that the polar extract was able to reduce by 60% (p<0,05) the enzymatic activity of sPLA2 Bj, regardless of the type of treatment applied, the reduction of the activity of sPLA2 Cd was reduced by 30% (p<0,05) when it was incubated for 30 min in presence of the extract. The modifications of the pathological effects caused by sPLA2s were evaluated by in vivo tests. The edematous action of sPLA2 Bj was reduced by 50% (p<0,05) when it was in presence of the extract, independent of incubation, since the same action for sPLA2 Cd was drastically reduced by the extract, however, after 60 min of administration of the agent inducer of inflammation, the myotoxic capacity with consequent release of creatine - kinase (CK) was modulated by the statement differently depending on the source of sPLA2, sPLA2 Bj to extract promoted a protective activity of CK release, because it reduced the release of enzyme, when applied intraperitoneally, 30 min before the offending agent, however, sPLA2 Cd showed reduced ability to release when CK was incubated with the extract for 30 min before application of the same muscle. Before the action of the extract on the sPLA2 induced inflammation was sought through the technique of polymerase chain reaction in real time (real time PCR), to determine the influence of the extract on the expression of genes involved in inflammation. We determined that Nf - Kb gene was the answer that although the presence of the extract is that the later is activated (or expressed), possibly because it was nuclear. Through these studies it was possible to determine the effect of these extracts on the acute inflammatory response induced by the action of sPLA2s from snake venoms / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

Orientadores: Sergio Marangoni, Luis Alberto Ponce Soto / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T10:22:36Z (GMT). No. of bitstreams: 1 Heleno_MauricioAurelioGomes_D.pdf: 8695465 bytes, checksum: 8ac64464abefb901cab1d4e5b6b96336 (MD5) Previous issue date: 2012 / Resumo: Uma grande variedade de toxinas provenientes de venenos animais tem sido largamente utilizada no estudo de mecanismos de ação e processos fisiológicos, sendo consideradas valiosas ferramentas moleculares. As serpentes peçonhentas expressam no veneno diversas proteínas muito estudadas e utilizadas clinicamente, apresentando diferentes graus de variabilidade inter e intraespecífica em suas composições e nos seus efeitos biológicos. O conhecimento obtido com os estudos destas moléculas tem grande importância clínica na compreensão dos processos fisiopatológicos envolvidos nos envenenamentos ofidicos e também acadêmico-tecnológica, devido à possibilidade do desenvolvimento de novos instrumentos moleculares utilizados na pesquisa e também de novos modelos moleculares para princípios ativos de drogas. Neste trabalho pesquisamos as atividades neurotóxica, miotóxica e inflamatória de uma PLA2 básica, D49, purificada do veneno da serpente Bothrops roedingeri após duas etapas cromatográficas, exclusão molecular em Sephadex G-75 e hidrofobicidade em HPLC de fase reversa em coluna 'mi'-Bondapak C-18. A BrTX-I apresentou massa molecular relativa em tomo de ~14 kDa (SDS-PAGE) e confirmada por espectrometria de massas (ESI-MS), em 14.358,69 Da. A análise da composição de aminoácidos da BrTX-I, revelou que esta é constituída aproximadamente por 120 resíduos aminoacídicos, com alto conteúdo de aminoácidos básicos e hidrofóbicos, resultando em um valor calculado de pI de 8,63. A presença de 14 resíduos de cisteína sugere a formação de sete pontes dissulfeto. A análise estrutural da BrTX-I foi realizada por ESI-MS e as regiões analisadas mostraram semelhança com outras PLA2 miotóxicas isoladas de venenos botrópicos. A BrTX-I apresentou alta atividade PLA2 e um comportamento tipo sigmoidal em baixas concentrações do substrato. Atividade PLA2 ótima da BrTX-I foi em pH 8,0 e temperatura de 37°C. A BrTX-I mostrou-se dependente de Ca2+ (mM) e na sua substituição por zn+2, Mn+2, Mg+2 e Cd+2 a atividade foi reduzida. O estudo da homologia sequencial da BrTX-I mostrou posições extremamente conservadas na molécula. Nas posições 1 e 2 há predominância da sequência de aminoácidos (DL), na posição 4 (Q). Uma das regiões altamente conservadas na sequência de aminoácidos das PLA2 é a alça de ligação ao cálcio, segmento ...YGCYCGXGG. Resíduos formando a alça de ligação ao cálcio e a rede catalítica da BrTX-I mostraram um alto grau de conservação, refletindo na manutenção da atividade. A região relacionada à atividade neurotóxica pré-sináptica (80-11 O) apresentou principalmente resíduos hidrofóbicos. Em preparações ex vivo, o veneno e a BrTX-I causaram rápido bloqueio da neurotransmissão na preparação biventer cervicis de pintainho de modo similar a outras Bothrops, sem alterar significativamente as respostas contraturantes à adição de AChe de KCl (5 e 20 'mi'g/mL), indicando atividade neurotóxica pré-sináptica. Em camundongos, a BrTX-I induziu miotoxicidade local, determinada pelo aumento nos níveis plasmáticos de CK e mostrou efeito pró-inflamatório analisado através da formação do edema de pata e liberação das citocinas IL-1 , IL-6 e TNF-'alfa'. Como BrTX-I produz um efeito inflamatório, a hidrólise de fosfolipídios pode ser relevante na fisiopatología do envenenamento / Abstract: A great variety of animal venom toxins has been widely used in the study of action mechanisms and metabolic processes, thus considered valuable molecular tools. Poisonous snakes contains in their venom several well studied and clinically used proteins, showing these venoms different intra or interspecific variability degrees in their composition and biological effects. The knowledge obtained with these molecules study, has a great clinical relevancy understanding pathophysiological process regarding snake envenomations, and also academic technological, due to the possibility to develop new molecular tools and new molecular models to study active principies of some drugs. ln this work, we study neurotoxic, myotoxic and inflammatory activities of BrTX-I, a basic PLA2, purified from Bothrops roendigeri snake venom after two chromatographic steps, using molecular exclusion chromatography (Sephadex G-75) and reverse phase HPLC on 'mi'-Bondapak C-18 column. BrTX-I showed relative molecular mass around 14 kDa (PAGE) and specific molecular mass of 14,358.69 Da was determined by ESl-MS mass spectrometry. The amino acid composition analysis showed that BrTX-I contains 120 aminoacidic residues with high content of basic and hydrophobic amino acids, resulting in a calculated pi value of 8. 63. The presence of 14 Cysteine residues, suggests the formation of seven dissulfide bonds. Structural analysis of BrTX-I PLA2, performed by ESI-MS showed high identity values when compared to other myotoxic PLA2, isolated from Bothrops snakes venoms. BrTX-I presented high PLA2 activity and showed a sigmoidal behavior at low substrate concentrations. The BrTX-I reached its maximal PLA2 activity at pH 8.0 and 37 °C. Maximum PLA2 activity required Ca2+ (mM) and substitution of Ca2+ by zn+2, Mn+2, Mg+2 or Cd+2 showed reduced enzymatic activity. Sequence homology studies of BrTX-I showed extremely conserved positions in the molecule. ln positions 1 and 2, there is a predominance of the amino acids sequence (DL), and in position 4 (Q). One of the highly conserved regions in the amino acid sequences of PLA2 is the Ca2+ -binding loop, segment ... YGCYCGXGG. Residues forming the Ca2+-binding loop and the catalytic network of BrTX-I PLA2 showed a high conservation grade, reflecting the non-decreased catalytic activity. The region related to the presynaptic neurotoxic activity (80-110), showed mainly the presence of hydrophobic residues. ln ex vivo studies, the whole venom and BrTX-I caused a fast blockade of the neuromuscular transmission in young chick biventer cervicis preparations m a similar way to other Bothrops species, without alters significantly the contractures induced by ACh and KCL at doses of 5 and 20 'mi'g/mL, respectively, indicating presynaptic neurotoxic activity. ln mice, BrTX-I induced local myotoxicity, determined by increase in CK serum leveis, and showed proinflammatory effects analyzed through edema-forming activity and citokines IL-1 , IL-6, and TNF'alpha' release. Once BrTX-I induces a strong pro-inflammatory effect, the enzymatic phospholipid hydrolysis may be relevant for envenomation pathophysiology / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Orientador: Sergio Marangoni / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T11:18:09Z (GMT). No. of bitstreams: 1 Ponce-Soto_LuisAlberto_D.pdf: 2824712 bytes, checksum: 7aeb46c8d2e647484f7c95e4df5cfb42 (MD5) Previous issue date: 2005 / Resumo: Através de metodologias otimizadas de purificação em HPLC, foram purificadas novas toxinas a partir do veneno total de Crotalus durissus collilineatus (V-1), Bothrops jararacussus PLA2 D49 Bj-V (isoforma de Bj-IV) obtida da fração BthTX-II e Bothrops alternatus PLA2 K49 Bt II-2; com um alto grau de pureza e homogeneidade molecular, sem perda da atividade biológica. A nova neurotoxina V-1 de Crotalus durissus collilineatus caracterizada físico-quimicamente como desprovida de atividade catalítica, ácida e com uma massa molecular de 9859.45 Da, uma única cadeia polipeptídica, induz uma facilitação contínua no modelo biológico nervo frênico diafragma isolado de camundongo e uma facilitação pronunciada seguida de um bloqueio súbito em biventer cervicis de pintainho, na junção neuromuscular. Os estudos estruturais, neurotóxicos e miotóxicos da PLA2 F6 do complexo crotoxina de Crotalus durissus collilineatus mostram que a PLA2 F6 precisa da crotapotina (estudos de re-associação) para potencializar seu efeito neurotóxico na preparação nervo frênico diafragma isolado de camundongo, mas a PLA2 F6 é capaz de induzir neurotoxicidade in vitro na ausência de crotapotina na preparação biventer cervicis de pintainho. Foram purificadas, a partir da fração BthTX-II, duas PLA2 D49 Bj-IV e V, sendo consideradas como isoformas devido ao fato de que compartilham de várias características físico-químicas, tais como: massa molecular, tempo de retenção na re-purificação em HPLC de fase reversa, análise de composição de aminoácidos, pI e estrutura primária, embora a isoforma Bj-V apresente algumas mutações com respeito à isoforma Bj-IV: W3 -> F3, Q4 -> E4, F5 -> W5, I16 -> N16 e G41 -> 41D; importantes para a atividade catalítica, os efeitos neurotóxicos in vitro foram mantidos e, quando foram inibidas cataliticamente por crotapotinas crotálicas de Crotalus durissus collilineatus (F3 e F4), continuaram diminuindo a resposta contrátil na junção neuromuscular nas preparações nervo frênico diafragma isolado de camundongo e biventer cervicis de pintainho, mostrando que são mais pronunciadas no músculo esquelético de camundongo. A nova toxina Bt II-2 purificada, com um alto grau de pureza e homogeneidade molecular, do veneno de Bothrops alternatus, foi caracterizada como uma PLA2 básica K49, em função das características físico-químicas, evidenciadas: massa de 13898.71 Da, possui caráter básico e uma alta homologia seqüencial na sua estrutura primária, quando comparada com outras PLA2 K49 procedentes de veneno de serpentes.Essa nova PLA2 K49 Bt II-2 revelou um potente efeito neurotóxico in vitro na preparação nervo frênico diafragma isolado de camundongo (1 µg/mL) pré-sináptico, como foi mostrado nos estudos de estímulo indireto via nervo e potencial de membrana ou repouso, diferentemente de qualquer PLA2 neurotóxica botrópica que, para causar um efeito neurotóxico, precisa de dosagem acima de 50 µg/mL. Outros perfis biológicos foram estudados para a Bt II-2, que mostrou uma miotoxicidade local ¿in vivo¿, citotoxicidade em mioblastos e miotubos C2C12, atividade inflamatória e letalidade, corroborando que se enquadra dentro da família de proteínas PLA2 homólogas K49, mas com características únicas nos estudos de neurotoxicidade in vitro. A reprodutibilidade da atividade biológica, através dos efeitos farmacológicos, só é possível com a utilização de frações quimicamente homogêneas que mantenham a integridade da função biológica. Essas frações são obtidas com metodologias de alta eficiência: HPLC, LC, espectrometria de massa, cujos resultados podem ser associados com sua atividade biológica, eliminando a subjetividade causada por veneno total ou frações impuras. Essa abordagem pode ser aplicada nos estudos bioquímicos, estrutura-função, fisiológicos e farmacológicos, podendo revelar mecanismos ainda desconhecidos na relação estrutura-função das PLA2 procedentes de veneno de serpentes. A presença de um sítio farmacológico distinto do sítio catalítico presente nas PLA2 pode ser usada como ferramenta molecular para o reconhecimento dos receptores de membrana desconhecidos em células ou tecidos / Abstract: Through optimized methodologies of purification in HPLC, new toxins were purified from total venom of Crotalus durissus collilineatus (V-1), Bothrops jararacussus PLA2 D49 Bj-V (Bj-IV isoform) obtained from the fraction BthTX-II and Bothrops alternatus PLA2 K49 Bt II-2; with a high level of purity and molecular homogeneity, with no loss of biological activity. The new neurotoxin V-1 of Crotalus durissus collilineatus, chemically and physically characterized as unproved of catalytic, acid activity and with a molecular mass of 9859.45 Da, an only polypeptide sequence, induces to a continual facilitation in the biological isolated mouse phrenic nerve diaphragm model of mouse and a pronunciated facility of a subital block in chick biventer cervicis preparation en, in the neuromuscular junction. The structural, neurotoxic and miotoxic studies on PLA2 F6 from crotoxin complex of Crotalus durissus collineatus show crotapotin is necessary (re-association studies) to increase its neurotoxic effect in nerve phrenic diaphragm isolated preparation of mouse, but the PLA2 F6 is able to induce neurotoxity in vitro in ausency of crotopatin in the preparation chick biventer cervicis. Two PLA2 D49 Bj-IV and V were purified from the fraction BthTX-II and considered isoform because they share several chemical and physical characteristics, such as molecular mass, retention time in re-purification in reverse phase HPLC, analyses of amino acid composition, pI and primary structure, although the Bj-V isoform shows some mutations regarding Bj-IV: W3 -> F3, Q4 -> E4, F5 -> W5, I16 -> N16 e G41 -> 41D isoform. Important for the catalytic activity, neurotoxic in vitro effects were maintained and, when catalytically inhibited by crotapotin crotalic of Crotalus durissus collilineatus (F3 e F4), they continued dimishing the contractile reply in neuromuscular junction in nerve phrenic diaphragm isolated preparations of mouse and preparation chick biventer cervicis, which shows they are more pronunciated in the skeleton muscle of the mouse. The new and purified BtII-2 toxin of venom of Bothrops alternatus, with a high level of purity and molecular homogeneity, was characterized as a PLA2 basic K49, because its chemical and physical evidenced characteristics, mass of 13898.71 Da, basic character and a high sequential homology in its primary structure, when it is compared with other PLA2 K49 from venom of serpents. This new PLA2 K49 Bt II-2 has revealed a potent neurotoxic effect in vitro in neuromuscular junction in nerve phrenic diaphragm isolated preparation of mouse (1 µg/mL) ante-synaptic, as it was proved by studies on indirect stimulus through nerve and potential of membrane or rest, differently of some neurotoxic botropic PLA2 that needs a dose above 50 µg/mL to cause a neurotoxic effect. Other biological profiles were studied for the Bt II-2, and the conclusion was a local myotoxic ¿living¿, a cytotoxic in myoblasts and myotubes C2C12, imflammatory and letality activity, showing they are fitted in the family of proteins PLA2 homologous K49, but with particular characteristics in studies on neurotoxic in vitro. The reprodubility of biological activity, through pharmacological effects, is just possible with the utilization of chemically homogenous fractions that maintain the integrity of biological function. These fractions were obtained with high efficient methodologies as HPLC, LC, and mass spectrometry, whose results may be associated with their biological activities, eliminating the subjectivity caused by total venom or impure fractions. This approximation may be applied in the biochemical studies, structure-function, physiological and pharmacological, and it may reveal still unknown mechanisms in the relation structure-function of PLA2 from the venom of serpents. The presence of a pharmacological site different of the catalytic one in the PLA2 may be used as a molecular instrument to the recognition of the receptors of unknown membranes in cells or tissues / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Prostaglandin (PG) E2 is an eicosanoid derived from the polyunsaturated twenty carbon fatty acid arachidonic acid (AA). PGE2 has physiological as well as pathophysiological functions and is known to be a key mediator of inflammatory responses. Formation of PGE2 is dependent upon the activities of three specific enzymes involved in the AA cascade; phospholipase A2 (PLA2), cyclooxygenase (COX) and PGE synthase (PGEs). Although the research within this field has been intense for decades, the regulatory mechanisms concerning the PGE2 synthesising enzymes are not completely established. PGE2 was investigated in human monocytes with or without lipopolysaccharide (LPS) pre-treatment followed by stimulation with calcium ionophore, opsonised zymosan or phorbol myristate acetate (PMA). Cytosolic PLA2a (cPLA2a) was shown to be pivotal for the mobilization of AA and subsequent formation of PGE2. Although COX-1 was constitutively expressed, monocytes required expression of COX-2 protein in order to convert the mobilized AA into PGH2. The conversion of PGH2 to the final product PGE2 was to a large extent due to the action of microsomal PGEs-1 (mPGEs-1). In addition, experiments with inhibitors of extracellular signal regulated kinase and p38 activation, indicated that phosphorylation of cPLA2α was markedly advantageous for the formation of PGE2. Ellagic acid, a natural polyphenolic compound found in fruits and nuts, was shown to inhibit stimuli induced release of PGE2 in human monocytes. The effect of ellagic acid was not due to a direct effect on the activities of the enzymes but rather to inhibition of the LPS-induced protein expression of COX-2, mPGEs-1 and cPLA2a.

As Fosfolipases A2 (PLA2) são enzimas que atuam desconstruindo membranas celulares, resultando em ácidos graxos e lisofosfolipidios, causando inflamação tecidual. Evidências indicam que serpentes possuem uma resistência natural devido a propriedades presentes no sangue, que inibem ações de proteínas presentes no veneno. Portanto, no presente trabalho foi isolado e caracterizado bioquimicamente e biologicamente o primeiro inibidor de PLA2 do tipo gama (&#947;PLI) do soro da serpente B. jararaca, denominado PLI_BJ. O inibidor de PLA2 foi isolado utilizando dois passos cromatográficos. O PLI_BJ mostrou, por SDS-PAGE, uma massa molecular aparente de 25 000 e 20 000 em condições redutoras e não redutoras, respectivamente. A sequência de aminoácidos parcial de PLI_BJ foi determinada por espectrometria de massa e corresponde a 72% e 68% de cobertura da sequência de aminoácidos de duas proteínas já descritas como PLI. O PLI_BJ mostrou também atividade inibitória satisfatória nos três testes realizados sugerindo um papel deste inibidor nos efeitos de envenenamento da serpente. / Phospholipases A2 (PLA2) are enzymes that act on cell membrane phospholipids resulting in fatty acids and lysophospholipids, deconstructing the cell wall causing tissue inflammation. Evidence indicates that snakes have natural resistance due to protective properties of blood that inhibits the action of proteins present in the venom. This study aimed to purify and characterize PLA2 inhibitors (PLI) from serum of the Bothrops jararaca snakes. PLA2 inhibitor was isolated using two chromatographic steps, and was named PLI_BJ. The purity of the PLI_BJ was confirmed by HPLC and SEC. The PLI_BJ showed, by SDS-PAGE, an molecular mass of 25,000 and 20,000 under reducing and non-reducing conditions, respectively. The partial amino acid sequence of PLI_BJ was determined by mass spectrometry and it corresponds to 72% and 68% of coverage of the amino acid sequence of two proteins already described as PLI. The PLI_BJ also showed satisfactory inhibitory activity in the three tests performed suggesting a role of this inhibitor in snake envenomation effects.

Secretory phospholipase A2 (sPLA2) is an important part of apoptosis and disposal of damaged and dying cells. However, healthy cells are not susceptible to attack by sPLA2. Recent studies have focused on membrane properties necessary to induce susceptibility in both artificial and biological membranes. Hydrolysis of phospholipids by sPLA2 requires at least two preliminary steps: first, adsorption of the enzyme to the cellular membrane, and second, movement of a phospholipid into the active site of the enzyme. We determined the effects of susceptibility on each of the two steps and determined the contributions changing the equilibrium constants have on susceptibility. The equilibrium constant for step one increased by a factor of 2 during susceptibility, while the equilibrium constant for step two increased by a factor of 4. The rise in the second equilibrium constant caused the majority of the change in hydrolysis rate seen during susceptibility; the influence of the first equilibrium constant is minimal. We confirmed these results with adsorption studies (assessment of the first step). We additionally found that sPLA2 has a high affinity for the cellular membrane and that only a small percentage (3-5%) of the membrane is covered when all adsorption sites are filled by the enzyme. We proposed a mathematical model describing the mechanism of action of sPLA2, and we were able to experimentally justify the assumptions made in the model.

Secretory phospholipase A2 (sPLA2) behaves differently toward the membranes of healthy cells compared to those of damaged or dying cells. The enzyme catalyzes rapid and sustained hydrolysis of compromised cells consistent with a simple catalytic mechanism. In contrast, when healthy cells are incubated with sPLA2, they become resistant to hydrolytic attack as manifest by three unusual observations: First, hydrolysis is transient and represents only a small fraction of the total membrane phospholipid content. Second, subsequent addition of sPLA2 fails to generate additional product. Third, the apparent potency of the enzyme to cause the membrane to be refractory is much greater than the potency for catalyzing hydrolysis. The mechanism responsible for this resistance has not yet been identified. Using Monte Carlo and direct analytical methods, we have developed a model capable of explaining all three of these observations. The model requires two salient elements: only a small pool of phospholipids in the healthy cell membrane is available for catalysis by sPLA2, and hydrolyzed phospholipids are re-acylated and restored very slowly to the accessible pool. The requirement for initial hydrolysis (as opposed to the simple physical presence of the enzyme as previously thought) was confirmed experimentally. Additional evidence has shown that the membrane does not remain permanently in its resistant state. Over time, the membrane resets to its original state. The model also predicts that total substrate, reacylation rate, and the return rate of phospholipids to the membrane should all be constant as enzyme concentration is varied. This prediction was tested by quantitative analysis of hydrolysis time courses at varied enzyme concentrations. Experiments with fluorescent probes, merocyanine 540 and laurdan suggest, that resistance may also involve physical changes to the membrane beyond the kinetic mechanisms hypothesized in the model.

A-B family of toxins consists of plant toxins such as ricin and bacterial toxins such as cholera. The A subunit is the enzymatic domain and the B subunit is the receptor binding domain. Commonly, these toxins bind to the target cell plasma membrane receptors through their B subunit followed by endocytosis and a transport to the endoplasmic reticulum (ER). Inside the ER, the A subunit dissociates from the rest of the toxin, unfolds and triggers the ER quality control mechanism of ER-associated degradation (ERAD). Most ERAD substrates are purged out of the ER into the cytosol for proteasomal degradation. However, the low content of lysine amino acid residues allows the toxin to evade polyubiquitination and subsequent proteasomal degradation. The toxin A subunit refolds into an active conformation in the cytosol, setting off downstream toxic events. In the first part of my thesis, the hypothesis was tested that inhibiting the unfolding of the toxin A subunit inside the ER will prevent ERAD activation, toxin export to the cytosol and intoxication. The chemical chaperones glycerol and sodium 4-phenyl butyrate (PBA) were used to inhibit the toxin A chain unfolding. In vitro biophysical experiments indicated that both chemical chaperones indeed stabilize the cholera toxin A subunit and prevent cytotoxicity. In case of ricin, both chaperones stabilized the toxin A chain but only glycerol prevented cytotoxicity. Additional experiments showed that PBA-treated ricin A chain is destabilized when exposed to anionic lipid membranes mimicking the properties of the ER membrane. In contrast, anionic lipid did not prevent ricin A chain stabilization by glycerol. This explains why glycerol but not PBA blocked ricin intoxication, as only glycerol stabilizes ricin A chain in the presence of ER membranes. Cholera toxin in contrast, remained either unaffected or slightly stabilized in presence of anionic lipids both in presence and absence of PBA. This shows that destabilization by anionic lipids is a toxin-specific rather than a general effect. In the second part of my thesis, the effect of inner leaflet of plasma membrane on the structure of cholera toxin A chain (CTA1) was studied. Since CTA1 refolds into an active conformation in the cytosol in association with unidentified host factors, I hypothesized that inner leaflet of the plasma membrane might play a role to stabilization and/or refolding of CTA1. CTA1 was shown to be a membrane interacting protein, and membranes mimicking lipid rafts had a significant stabilizing effect on its structure. Lipid rafts helped in the regaining of the tertiary and secondary structure of CTA1, while non-raft lipids had a smaller stabilizing effect on CTA1 structure. In the next part of my thesis, I studied the effect of membrane binding on the structure and function of human pancreatic phospholipase A₂ (PLA₂). Lipid thermal phase transition was found to have a dramatic effect on PLA₂ activity. It was also established that although membrane binding and insertion was essential for of PLA₂ activity, lipid structural heterogeneity was more important than the depth of membrane insertion for enzyme activation. Most importantly, significant changes in PLA₂ secondary and tertiary structures were identified that evidently contribute to the interfacial activation of PLA₂. Overall, we conclude that the function of membrane binding enzymes can be significantly modulated via conformational changes induced by interactions with membranes. Thus, we have elucidated various roles of membrane lipids from unfolding and refolding to activation and modulation of membrane binding enzymes. Physical properties of lipids help in regulating various aspects of protein structure and function and their analysis helped us in appreciating the influence wielded by the membrane lipids in the enzyme's surrounding environment.

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