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.

Orientador: Marcos Hikari Toyama / Resumo: Os acidentes ofídicos foram recentemente incorporados na lista de doenças tropicais negligenciadas pela Organização Mundial de Saúde (OMS). No Brasil, uma parcela significativa destes acidentes (10%) é ocasionada pela serpente Crotalus durissus terrificus (Cdt), popularmente conhecida como cascavel. Dentre os componentes do veneno, a fosfolipase A2 secretória (sPLA2) representa aproximadamente 35% da massa do veneno seco. A Cdt sPLA2 responde pela atividade mais importante e clinicamente significativa que inicia com o processo inflamatório logo após o contato com a peçonha. Quando não tratada, a peçonha pode fazer com que o indivíduo sofra com insuficiência renal aguda, a qual não é neutralizada em curto espaço de tempo, nem mesmo pelo antiveneno específico. O processo inflamatório em diversas doenças humanas, está altamente relacionado com a formação de altas quantidades de espécies reativas de oxigênio e nitrogênio (EROs e ERNs), conhecido como explosão oxidativa/nitrosativa. Este processo é combatido por moléculas de baixo peso molecular como (vitaminas D e E, e glutationa), como também por enzimas antioxidantes, como a catalase (Cat), superóxido dismutase (Sod) e peroxirredoxinas (Prx). Já foi demonstrado que as sPLA2 humanas compartilham grande similaridades bioquímicas e farmacológicas com seus homólogos de serpentes e são capazes de induzir a formação de EROs e ERNs em células de mamíferos, mas nenhum estudo até o presente momento abordou de forma sistemática danos em... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Snake envenomation have recently been added to the list of tropical neglected diseases by the World Health Organization (WHO). In Brazil, a significant portion of ophidian accidents (~ 10%) is caused by the Crotalus durissus terrificus (Cdt) snake, popularly known as rattlesnake. Among the components of the venom, secretory phospholipase A2 (sPLA2) accounts for approximately 35% of the mass of the dry venom. Cdt sPLA2 accounts for the most important clinical damages and to the ingflammmatory proccess. When untreated, venomation can cause acute renal failure, which is not neutralized in a short time, not even by the specific antivenom. The inflammatory process, which is very well characterized in several human diseases, is highly related to the formation of high amounts of reactive oxygen and nitrogen species (ROS and RNS), known as oxidative / nitrosative burst. This process is counteracted by low molecular weight molecules such as vitamins D and E and glutathione, as well as antioxidant enzymes such as peroxiredoxins (Prx). human sPLA2 share high biochemical and pharmacological similarities with their snake homologs and are able to induce the formation of ROS and RNS in mammalian cells, but no study to date has systematically addressed damage to biomolecules carried out by ROS and RNS and the expression of peroxiredoxins in response to sPLA2 administration of snakes. We have recently shown that the administration of polyphenolic extracts of Laguncularia racemosais able to in... (Complete abstract click electronic access below) / Mestre

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No. of bitstreams: 1 ie_r_me_svic.pdf: 2864379 bytes, checksum: 0b7eb6759926f7f64a9bfde5500083ea (MD5) Previous issue date: 2018-02-22 / Outra / Os acidentes ofídicos foram recentemente incorporados na lista de doenças tropicais negligenciadas pela Organização Mundial de Saúde (OMS). No Brasil, uma parcela significativa destes acidentes (10%) é ocasionada pela serpente Crotalus durissus terrificus (Cdt), popularmente conhecida como cascavel. Dentre os componentes do veneno, a fosfolipase A2 secretória (sPLA2) representa aproximadamente 35% da massa do veneno seco. A Cdt sPLA2 responde pela atividade mais importante e clinicamente significativa que inicia com o processo inflamatório logo após o contato com a peçonha. Quando não tratada, a peçonha pode fazer com que o indivíduo sofra com insuficiência renal aguda, a qual não é neutralizada em curto espaço de tempo, nem mesmo pelo antiveneno específico. O processo inflamatório em diversas doenças humanas, está altamente relacionado com a formação de altas quantidades de espécies reativas de oxigênio e nitrogênio (EROs e ERNs), conhecido como explosão oxidativa/nitrosativa. Este processo é combatido por moléculas de baixo peso molecular como (vitaminas D e E, e glutationa), como também por enzimas antioxidantes, como a catalase (Cat), superóxido dismutase (Sod) e peroxirredoxinas (Prx). Já foi demonstrado que as sPLA2 humanas compartilham grande similaridades bioquímicas e farmacológicas com seus homólogos de serpentes e são capazes de induzir a formação de EROs e ERNs em células de mamíferos, mas nenhum estudo até o presente momento abordou de forma sistemática danos em biomoléculas efetuados por EROs e ERNse a expressão de proteínas antioxidantes em resposta a administração de sPLA2 de serpentes. Recentemente demonstramos que a administração de extratos de Laguncularia racemosa (mangue branco) capazes de inibir a ação da trombina, entretanto não foram efetuadas avaliações se a aplicação do extrato de L. racemosa frente a Cdt sPLA2. Também e importante salientar que apesar de existir inúmeros trabalhos de caracterização bioquímica, farmacológica e estrutural de sPLA2 de serpentes até o presente momento poucos trabalhos tiveram como objetivo sua produção de forma recombinante. Este ponto é importante pois a manipulação gênica destas proteínas, através de mutações sitio dirigidas, podem auxiliar a identificar resíduos envolvidos na catálise, levando a uma melhor compreensão dos mecanismos funcionais, abrindo caminho para novas abordagens terapêuticas para o tratamento do envenenamento por serpentes. Adicionalmente, a enzima sPLA2 de origem animal é utilizada em processos biotecnológicos e possui importância econômica. Este trabalho teve dois grandes objetivos: 1) investigar a existência de danos oxidativos, a expressão de proteínas antioxidantes em resposta a administração de sPLA2 de Crotalus durissus terrificus (Cdt sPLA2), bem com avaliar possíveis efeitos protetores da administração de extratos butanólico e de acetato de etila extraídos de folhas de L. racemosa após a inoculação da Cdt sPLA2 na fase tardia da edemaciação; 2) Obtenção de sPLA2 recombinante de Cdt (Cdt sPLA2r) visando fornecer subsídios para sua manipulação genética e maior entendimento de seu funcionamento. Com relação ao primeiro objetivo nossos resultados indicam que os extratos de L. racemosa não são capazes de inibir o processo de edemaciação induzido por Cdt sPLA2. A investigação de processos indicadores de estresse oxidativo como oxidação de sulfidrilas, carbonilação proteica e peroxidação lipídica não idicaram a existência de estresse oxidativo na fase tardia do processo de edemaciação ocasionado por Cdt sPLA2. Tambem avaliamos os níveis de expressão e modificação das proteínas antioxidantes Sod, Cat e Prx2 por western blot, e os resultados também revelaram que os níveis da enzima foram muito similares independente da administração de Cdt sPLA2 ou Cdt sPLA2 com extratos de L. racemosa. Em conjunto os resultados indicam fortemente que não ocorre estresse oxidativo na fase tardia do processo da edemaciação por Cdt sPLA2.No caso do segundo objetivo foi possível clonar, expressar e purificar a enzima recombinante Cdt sPLA2 (Cdt sPLA2r). Análises da atividade de fosfolipase revelaram que Cdt sPLA2r possuipadrão de atividade Michaeliana ao passo que a enzima purificada do veneno possui perfil alostérico. Adicionalmente a velocidade inicial (V0) de Cdt sPLA2r(10.8 × 10-1 µM/s) foi maior que para a enzima nativa (7.1 × 10-1 µM/s). A expressão de Cdt sPLA2r abre caminhos para investigações envolvendo mutações sítio dirigidas para melhor entendimento da enzima, a qual também possui importância biotecnológica. Em conjunto os resultados apresentados neste trabalho representam avanços na compreensão do processo de toxicológico envolvido na ação de Cdt sPLA2 e de sua expressão recombinante. / Snake envenomation have recently been added to the list of tropical neglected diseases by the World Health Organization (WHO). In Brazil, a significant portion of ophidian accidents (~ 10%) is caused by the Crotalus durissus terrificus (Cdt) snake, popularly known as rattlesnake. Among the components of the venom, secretory phospholipase A2 (sPLA2) accounts for approximately 35% of the mass of the dry venom. Cdt sPLA2 accounts for the most important clinical damages and to the ingflammmatory proccess. When untreated, venomation can cause acute renal failure, which is not neutralized in a short time, not even by the specific antivenom. The inflammatory process, which is very well characterized in several human diseases, is highly related to the formation of high amounts of reactive oxygen and nitrogen species (ROS and RNS), known as oxidative / nitrosative burst. This process is counteracted by low molecular weight molecules such as vitamins D and E and glutathione, as well as antioxidant enzymes such as peroxiredoxins (Prx). human sPLA2 share high biochemical and pharmacological similarities with their snake homologs and are able to induce the formation of ROS and RNS in mammalian cells, but no study to date has systematically addressed damage to biomolecules carried out by ROS and RNS and the expression of peroxiredoxins in response to sPLA2 administration of snakes. We have recently shown that the administration of polyphenolic extracts of Laguncularia racemosais able to inhibit the action of thrombin, however, were not evaluated the action of L. racemosa extractsadministration combat the deleterious effects of the Cdt sPLA2. It is also important to point out that although there are numerous biochemical, pharmacological and structural characterizations of sPLA2 from snakes, at the presene, few studies have aimed at its production by recombinant techniques. This is very important since the genetic manipulation of these proteins through site-directed mutations can help identify residues involved in catalysis, leading to a better understanding of the functional mechanisms, which may in future be reflected in new therapeutic approaches. Additionally, these enzymes are used in biotechnological processes.This work had two main objectives: 1) to investigate the existence of oxidative damages, the expression of antioxidant proteins in response to sPLA2 administration of Crotalus durissus terrificus (Cdt sPLA2), and to evaluate possible protective effects of the administration of butanolic and acetate extracts of ethylene extracted from L. racemosa leaves after inoculation of the Cdt sPLA2 at the late stage of edema; 2) the production of recombinant Cdt sPLA2 (Cdt sPLA2r) to provide subsidies for its genetic manipulation aiming a better understanding of molecular aspects of the enzyme function as also its applicability in biotechnological processes. Regarding the first objective, our results indicate that extracts of L. racemosawere not able to inhibit the process of the edema induced by Cdt sPLA2. The investigation of oxidative stress markers such as sulfhydryl oxidation, protein carbonylation and lipid peroxidation did not identify the existence of oxidative stress in the late phase of the edemaprocess caused by Cdt sPLA2. We also evaluated levels of expression and modification of the antioxidant proteins Sod, Cat and Prx2 by western blot, and the results also revealed that enzyme levels were very similar regardless of the administration of Cdt sPLA2 or Cdt sPLA2 with extracts of L. racemosa. Together the results strongly indicate that oxidative stress does not occur in the late phase of the edema caused by Cdt sPLA2. In the case of the latter objectives it was possible to clone, express and purify the recombinant enzyme Cdt sPLA2 (Cdt sPLA2r). Analysis of the phospholipase activity revealed that Cdt sPLA2r has a Michaelian environment activity whereas the enzyme purified from the venom has an allosteric profile. In addition, the initial velocity (V0) of the Cdt sPLA2r (10.8 × 10-1 μM / s) was higher than for the native enzyme (7.1 × 10-1 μM / s). The expression of Cdt sPLA2r opens pathways for investigations involving site directed mutations to a better understanding of the enzyme, which also has biotechnological importance. Together the results presented in this work represent advances in the understanding of the toxicological process involved in the action of Cdt sPLA2 and its recombinant expression.

Orientador: Marcos Hikari Toyama / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T09:30:17Z (GMT). No. of bitstreams: 1 Cotrim_CamilaAparecida_M.pdf: 24882799 bytes, checksum: f5ac35590f5619d941a17e8aa3f11bb4 (MD5) Previous issue date: 2011 / Resumo: Nas últimas décadas, o uso de compostos naturais como flavonóides e sapogeninas tem sido largamente difundidos na indústria farmacêutica devido suas atividades antiinflamatórias, prevenção de câncer e de doenças cardiovasculares. Neste trabalho, foi avaliado o efeito da quercetina (flavonóide) e hecogenina (sapogenina), sobre as atividades catalítica e farmacológicas de uma isoforma de fosfolipase A2 secretória (sPLA2) de Crotalus durissus terrificus em duas situações: pré-incubado e co-incubado. Na préincubação, a sPLA2 pura foi incubada com os compostos e os resultados indicam modificações estruturais na estrutura secundária como evidenciada pelas análise de dicroísmo circular. Os dois compostos foram capazes de diminuir a atividade enzimática, porém, somente a quercetina foi capaz de diminuir a mionecrose. Nenhum dos compostos apresentou efeito sobre a formação de edema. Na co-incubação, três concentrações diferentes dos compostos foram misturados com sPLA2 e imediatamente testados. A coincubação mostrou uma menor ação sobre atividade catalítica quando comparado com a préincubação, sugerindo a importância da incubação dos compostos com a proteína. Entretanto, a co-incubação mostrou melhor efeito sobre as atividades farmacológicas (edema e miotoxidade). Os resultados obtidos sugerem a existência de dois sítios farmacológicos distintos, um relacionado com sítio enzimático e outro distinto dessa atividade. Além disso, estudos de docking realizados entre a sPLA2 e a quercetina mostraram a existência de ligações de hidrogênio, interações polares e hidrofóbicas, sugerindo que outros flavonóides com estruturas similares podem se ligar à sPLA2. Além de avaliar o efeito da quercetina sobre a sPLA2 de Crotalus durissus terrificus foi avaliado o efeito sobre uma sPLA2 cataliticamente inativa (Lys49-sPLA2) de Bothrops pirajai. O resultado de dicroísmo circular não apresentou modificações em nível de estrutura secundária, entretanto, estudo de estabilidade mostrou que a quercetina leva a uma maior estabilidade térmica da estrutura proteica frente a altas temperaturas, tornando o processo de desnaturação reversível. Apesar da ausência de atividade catalítica, Lys49-sPLA2 apresentou formação de edema, miotoxidade e uma baixa agregação plaquetária. Entretanto, nenhuma das atividades farmacológicas foi inibida significativamente pela quercetina. Os resultados apresentados mostram que o uso de compostos fenólicos são uma boa iniciativa para estudar e melhor entender as ações de sPLA2 purificadas do veneno de serpente. / Abstract: In the last decades, the use of natural compounds, such as flavonoids and sapogenins has been applied to various pharmaceutical industries due their anti-inflammatory, câncer preventive and cardiovascular protective activities. In this study was evaluated the effect of quercetin (flavonoid) and hecogenin (sapogenin) on the catalytic and pharmacological activity of a secretoy phospholipase A2 from Crotalus durissus terrificus in two different situations: pre-incubated and co-incubated. In the pre-incubation situation, native sPLA2 was incubated with the compounds and the results have shown structural modifications in secondary structure as evidenced through circular dicroism. Both compounds were able to decrease catalytic activity, however, just quercetin was able to decrease myonecrosis. None of the compounds have shown effects on the oedema formation. In the co-incubation, three different concentrations of both compounds were mixed with sPLA2 and immediately tested. Co-incubation has shown lesser effect on catalytic activity than pre-incubation, suggesting an important role of incubation process. Nevertheless, co-incubation presented better effects in pharmacological activities (oedema and myotoxic). These results suggest the existence of two pharmacological sites in the protein, one that is correlated with the enzymatic site and another that is distinct from it. In addition, molecular docking studies between sPLA2 and quercetin showed the existence of hydrogen-bonded, polar interactions and hydrophobic interactions, suggesting that other flavonoids with similar structures could bind to sPLA2. Besides to evaluate the effect of quercetin on a Crotalus durissus terrificus sPLA2, was also evaluated the effect of this compound on a sPLA2 inactive catalytically (Lys49-sPLA2) from Bothrops pirajai. Dicroism circular results did not show modifications in secondary structure, however, thermal stability study have shown that quercetin is able to increase the stability of sPLA2 in high temperatures, in addition to become the denaturation a reversive process. Despite of the lack of catalytic activity, Lys49-sPLA2 has shown oedema formation, myotoxic and a low platelet aggregation. Although none of pharmacological activities was significantly abolish by quercetin. The results have shown that the use of phenolics compounds are a good point to study and better understand the actions of sPLA2 purified from venom snake. / Mestrado / Bioquimica / Mestre Biologia Funcional e Molecular

Secretory phospholipase A2 hydrolyzes phospholipids at a lipid-water interface, resulting in pro-inflammatory products being released from cell membranes. Healthy cells are resistant to cleavage by this enzyme, but apoptotic cells become susceptible to its activity. Only bilayers with certain characteristics are able to be hydrolyzed. Most recently, studies in this lab have emphasized the idea that the biophysical state of the bilayer (in terms of lipid order, spacing, and fluidity) is relevant in determining the probability of one phospholipid escaping the membrane to be hydrolyzed. Prior to this study, it had been shown that apoptotic cells undergo biophysical alterations that weaken inter-lipid interactions early in apoptosis. The purpose of this dissertation was to examine these changes in more detail, define them more clearly on the molecular level, and suggest possible mechanisms responsible for their occurrence. First, the role of increased membrane permeability in susceptibility to the phospholipase was investigated. S49 cells were treated with ionomycin or apoptotic agents and assayed for merocyanine 540 staining of the membrane and membrane permeability to a vital dye. Human group X and snake venom isoforms were active towards all treated cells, but human groups V and IIa only hydrolyzed cells that were moderately permeable to the vital dye. Different isoforms must then be sensitive to different membrane properties. Second, the role of membrane oxidation in cell membrane vulnerability to the phospholipase (specifically human group IIa) was tested. The temporal onset of lipid peroxidation was assayed during apoptosis. This correlated with the onset of susceptibility to the IIa isoform. Direct oxidizers were then used to verify this result in isolation from other apoptotic membrane changes. Third, biophysical alterations during thapsigargin-induced apoptosis were examined using TMA-DPH and Patman. Data from these probes in artificial bilayers undergoing phase transitions were used to quantify the decrease in interlipid interactions and predict a 50 -- 100-fold increase in the probability of phospholipid protrusions. Patman equilibration kinetics also revealed more molecular detail about the biophysical changes related to susceptibility. Finally, temperature- and ionomyin-induced alterations in membrane properties were compared. Both increased fluidity, but only ionomycin caused susceptibility. Patman equilibration kinetic analysis could distinguish responsible membrane properties. Actin fragmentation during apoptosis or calcium loading is proposed as the mechanism.