Littérature scientifique sur le sujet « Phospholipase A2 (sPLA2) »

The sensitivity of different phospholipase A2 (PLA2)-active fractions eluted from cation-exchange chromatography to para-bromophenacylbromide (pBPB), Ca2+-EGTA, DTT, heat, and H2SO4 indicates that human cultured retinal pigment epithelial (hRPE) cells probably contain two different intracellular PLA2 enzymes. Control experiments using "back-and-forth" thin-layer chromatography confirmed that, in our assay conditions, the generation of free fatty acids originated solely from PLA2 activity. Together with immunoblot experiments where no cross-reactivity was observed between the hRPE cytosolic PLA2 enzymes and several antisera directed against secretory PLA2s (sPLA2s) and cytosolic PLA2 (cPLA2), these findings suggest that intracellular hRPE PLA2s are different from well-known sPLA2s, cPLA2, and Ca2+-independent PLA2s. We also report an additional hRPE-PLA2 enzyme that is secreted and that exhibits sensitivity to pBPB, Ca2+-EGTA, DTT, heat, and H2SO4, which is characteristic of sPLA2 enzymes. This approximately 22-kDa PLA2 cross-reacted weakly with an antiserum directed against porcine pancreatic group I sPLA2 but strongly with an antiserum directed against N-terminal residues 1-14 of human synovial group II sPLA2, suggesting that this extracellular enzyme is a member of the sPLA2 class of enzymes. We thus conclude that there are three distinct PLA2 enzymes in cultured hRPE cells, including two novel intracellular PLA2s and a 22-kDa secreted sPLA2 enzyme.Key words: phospholipase A2, retinal pigment epithelium, characterization.

Abstract Secreted phospholipase A2 (sPLA2) regulates a variety of cellular functions. The present investigation was undertaken to elucidate the potential role of sPLA2 in endothelial cell (EC) migration. Bovine aortic endothelial cells (BAECs) exposed to sPLA2 placed in the lower compartment of a modified Boyden chamber displayed increased migration compared to cells exposed to vehicle. The effect of sPLA2 on EC migration was time and dose dependent. Migration of BAECs was observed at 30 minutes, increased over 1 to 2 hours, and declined thereafter. At 2 hours of stimulation, sPLA2 (0.01-2 μmol/L) induced 1.2- to 3-fold increased cell migration compared with media alone. Among the different sPLA2s tested, bee venom, Naja naja, and porcine and human pancreatic PLA2s all evoked a migratory response in ECs. Moreover, human synovial fluid, obtained from patients with arthritis and containing sPLA2 activity, induced EC migration. Migration of ECs was significantly reduced after exposure to a catalytic site mutant of pancreatic sPLA2with decreased lipolytic activity as compared to wild-type sPLA2. Similarly, pretreatment of human synovial fluid withp-bromophenacyl bromide, an irreversible inhibitor of sPLA2, markedly decreased the ability of human synovial fluid to stimulate EC migration. Moreover, migration of ECs was stimulated on exposure to hydrolytic products of sPLA2activity including arachidonic acid, lysophosphatidic acid, and lysophosphatidylcholine. These findings suggest that sPLA2plays a physiologic role in induction of EC migration. Moreover, the effects of sPLA2 on EC migration are mediated, at least in part, by its catalytic activity.

Secreted phospholipase A2 (sPLA2) regulates a variety of cellular functions. The present investigation was undertaken to elucidate the potential role of sPLA2 in endothelial cell (EC) migration. Bovine aortic endothelial cells (BAECs) exposed to sPLA2 placed in the lower compartment of a modified Boyden chamber displayed increased migration compared to cells exposed to vehicle. The effect of sPLA2 on EC migration was time and dose dependent. Migration of BAECs was observed at 30 minutes, increased over 1 to 2 hours, and declined thereafter. At 2 hours of stimulation, sPLA2 (0.01-2 μmol/L) induced 1.2- to 3-fold increased cell migration compared with media alone. Among the different sPLA2s tested, bee venom, Naja naja, and porcine and human pancreatic PLA2s all evoked a migratory response in ECs. Moreover, human synovial fluid, obtained from patients with arthritis and containing sPLA2 activity, induced EC migration. Migration of ECs was significantly reduced after exposure to a catalytic site mutant of pancreatic sPLA2with decreased lipolytic activity as compared to wild-type sPLA2. Similarly, pretreatment of human synovial fluid withp-bromophenacyl bromide, an irreversible inhibitor of sPLA2, markedly decreased the ability of human synovial fluid to stimulate EC migration. Moreover, migration of ECs was stimulated on exposure to hydrolytic products of sPLA2activity including arachidonic acid, lysophosphatidic acid, and lysophosphatidylcholine. These findings suggest that sPLA2plays a physiologic role in induction of EC migration. Moreover, the effects of sPLA2 on EC migration are mediated, at least in part, by its catalytic activity.

Phospholipase A2 enzymes (PLA2s) catalyze the hydrolysis of glycerophospholipids at their sn-2 position releasing free fatty acids and lysophospholipids. Mammalian PLA2s are classified into several categories of which important groups include secreted PLA2s (sPLA2s) and cytosolic PLA2s (cPLA2s) that are calcium-dependent for their catalytic activity and calcium-independent cytosolic PLA2s (iPLA2s). Platelet-activating factor acetylhydrolases (PAF-AHs), lysosomal PLA2s, and adipose-specific PLA2 also belong to the class of PLA2s. Generally, cPLA2 enzymes are believed to play a major role in the metabolism of arachidonic acid, the iPLA2 family to membrane homeostasis and energy metabolism, and the sPLA2 family to various biological processes. The focus of this review is on recent research developments in the sPLA2 field. sPLA2s are secreted enzymes with low molecular weight (with the exception of GIII sPLA2), Ca2+-requiring enzymes with a His-Asp catalytic dyad. Ten enzymatically active sPLA2s and one devoid of enzymatic activity have been identified in mammals. Some of these sPLA2s are potent in arachidonic acid release from cellular phospholipids for the biosynthesis of eicosanoids, especially during inflammation. Individual sPLA2 enzymes exhibit unique tissue and cellular localizations and specific enzymatic properties, suggesting their distinct biological roles. Recent studies indicate that sPLA2s are involved in diverse pathophysiological functions and for most part act nonredundantly.

Treating osteoarthritis (OA) remains a major clinical challenge. Despite recent advances in drug discovery and development, no disease-modifying drug for knee OA has emerged with any notable clinical success, in part, due to the lack of valid and responsive therapeutic targets and poor drug delivery within knee joints. In this work, we show that the amount of secretory phospholipase A2 (sPLA2) enzyme increases in the articular cartilage in human and mouse OA cartilage tissues. We hypothesize that the inhibition of sPLA2 activity may be an effective treatment strategy for OA. To develop an sPLA2-responsive and nanoparticle (NP)–based interventional platform for OA management, we incorporated an sPLA2 inhibitor (sPLA2i) into the phospholipid membrane of micelles. The engineered sPLA2i-loaded micellar NPs (sPLA2i-NPs) were able to penetrate deep into the cartilage matrix, prolong retention in the joint space, and mitigate OA progression. These findings suggest that sPLA2i-NPs can be promising therapeutic agents for OA treatment.

Among the phospholipase A2 (PLA2) superfamily, the secreted PLA2 (sPLA2) family contains 11 mammalian isoforms that exhibit unique tissue or cellular distributions and enzymatic properties. Current studies using sPLA2-deficient or -overexpressed mouse strains, along with mass spectrometric lipidomics to determine sPLA2-driven lipid pathways, have revealed the diverse pathophysiological roles of sPLA2s in various biological events. In general, individual sPLA2s exert their specific functions within tissue microenvironments, where they are intrinsically expressed through hydrolysis of extracellular phospholipids. Recent studies have uncovered a new aspect of group IIA sPLA2 (sPLA2-IIA), a prototypic sPLA2 with the oldest research history among the mammalian PLA2s, as a modulator of the gut microbiota. In the intestine, Paneth cell-derived sPLA2-IIA acts as an antimicrobial protein to shape the gut microbiota, thereby secondarily affecting inflammation, allergy, and cancer in proximal and distal tissues. Knockout of intestinal sPLA2-IIA in BALB/c mice leads to alterations in skin cancer, psoriasis, and anaphylaxis, while overexpression of sPLA2-IIA in Pla2g2a-null C57BL/6 mice induces systemic inflammation and exacerbates arthritis. These phenotypes are associated with notable changes in gut microbiota and fecal metabolites, are variable in different animal facilities, and are abrogated after antibiotic treatment, co-housing, or fecal transfer. These studies open a new mechanistic action of this old sPLA2 and add the sPLA2 family to the growing list of endogenous factors capable of affecting the microbe–host interaction and thereby systemic homeostasis and diseases.

Hydrolysis of surfactant-associated phospholipids by secretory phospholipases A2 is an important potential mechanism for surfactant dysfunction in inflammatory lung diseases. In these conditions, airway secretory phospholipase A2(sPLA2) activity is increased, but the type of sPLA2 and its impact on surfactant function are not well understood. We examined in vitro the effect of multiple secretory phospholipases A2 on surfactant, including their ability to 1) release free fatty acids, 2) release lysophospholipids, and 3) increase the minimum surface tension (γmin) on a pulsating bubble surfactometer. Natural porcine surfactant and Survanta were exposed to mammalian group I (recombinant porcine pancreatic) and group II (recombinant human) secretory phospholipases A2. Our results demonstrate that mammalian group I sPLA2 hydrolyzes phosphatidylcholine (PC), producing free fatty acids and lysophosphatidylcholine, and increases γmin. In contrast, mammalian group II sPLA2 demonstrates limited hydrolysis of PC and does not increase γmin. Group I and group II secretory phospholipases A2 from snake venom hydrolyze PC and inhibit surfactant function. In summary, mammalian secretory phospholipases A2 from groups I and II differ significantly from each other and from snake venom in their ability to hydrolyze surfactant-associated PC.

Secreted phospholipase A2 (sPLA2) molecules constitute a family of proteins that are involved functionally in many biological processes. In particular, they participate in diverse pathophysiological settings as enzymes that release free fatty acids and lysophospholipids from phospholipids in biological membranes, or as ligands for various cellular receptors. In this review the confirmed or expected functions of sPLA2s in the mammalian immune system are surveyed. Some of the twelve mammalian sPLA2 molecules constitute part of the so-called innate immune system by virtue of their antibacterial, antiviral and antifungal activities. They are also involved in acute inflammation, a protective reaction of the body to infection or injury. The acute inflammation sometimes escapes regulation, becomes chronic and can evolve into a severe pathology. One or more types of sPLA2 are involved in asthma, rheumatoid arthritis, sepsis, atherosclerosis, myocardial infarction, Crohn’s disease, ulcerative colitis and cancer. sPLA2s are thus important therapeutic targets as well as biotherapeutic molecules. Improving the selectivity of inhibitors of sPLA2s to be able to target a particular sPLA2 could therefore be one of the most important tasks for future research.

Among several different types of phospholipase A2 (PLA2), cytosolic PLA2 (cPLA2)α and group IIA (IIA) secretory PLA2 (sPLA2) have been studied intensively. To determine the discrete roles of cPLA2α in platelets, we generated two sets of genetically engineered mice (cPLA2α−/−/sPLA2-IIA−/− and cPLA2α−/−/sPLA2-IIA+/+) and compared their platelet function with their respective wild-type C57BL/6J mice (cPLA2α+/+/sPLA2-IIA−/−) and C3H/HeN (cPLA2α+/+/sPLA2-IIA+/+). We found that cPLA2α is needed for the production of the vast majority of thromboxane (TX)A2 with collagen stimulation of platelets. In cPLA2α-deficient mice, however, platelet aggregation in vitro is only fractionally decreased because small amounts of TX produced by redundant phospholipase enzymes sufficiently preserve aggregation. In comparison, adenosine triphosphate activation of platelets appears wholly independent of cPLA2α and sPLA2-IIA for aggregation or the production of TX, indicating that these phospholipases are specifically linked to collagen receptors. However, the lack of high levels of TX limiting vasoconstriction explains the in vivo effects seen: increased bleeding times and protection from thromboembolism. Thus, cPLA2α plays a discrete role in the collagen-stimulated production of TX and its inhibition has a therapeutic potential against thromboembolism, with potentially limited bleeding expected.

Pulmonary surfactant's complex mixture of phospholipids and proteins reduces the work of breathing by lowering alveolar surface tension during respiration. One mechanism of surfactant damage appears to be the hydrolysis of phospholipid by phospholipases activated in the inflamed lung. Humans have several candidate secretory phospholipase A2 (sPLA2) enzymes in lung cells and infiltrating leukocytes that could damage extracellular surfactant. We considered two mechanisms of surfactant disruption by five human sPLA2s, including generation of lysophospholipids and the depletion of specific phospholipids. All five sPLA2s studied ultimately caused surfactant dysfunction. Each enzyme exhibited a different pattern of hydrolysis of surfactant phospholipids. Phosphatidylcholine, the major phospholipid in surfactant and the greatest potential source for generation of lysophospholipids, was susceptible to hydrolysis by group IB, group V, and group X sPLA2s, but not group IIA or IID. Group IIA hydrolyzed both phosphatidylethanolamine and phosphatidylglycerol, whereas group IID was active against only phosphatidylglycerol. Thus, with groups IB and X, the generation of lysophospholipids corresponded with surfactant dysfunction. However, hydrolysis of and depletion of phosphatidylglycerol had a greater correlation with surfactant dysfunction for groups IIA and IID. Surfactant dysfunction caused by group V sPLA2 is less clear and may be the combined result of both mechanisms.

Les phospholipases A catalysent sélectivement l'hydrolyse de la liaison ester des glycérophospholipides dans la position sn-1 (PLA1) et sn-2 (PLA2), libérant des acides gras et des lysophospholipides. Nous avons identifié des PLAs à partir des champignons/actinomycètes isolés des environnements du Mexique. Dans la 1ère partie, une méthode colorimétrique à haut débit pour la détection générale de l'activité PLA (cHTS-PLA) avec la PC comme substrat et le rouge crésol a été développée. L'analyse rRNA 16S des souches productrices des PLAs a démontrant qu'elles appartiennent aux genres Streptomyces et Aureobasidium. La production des PLAs a été mise en œuvre utilisant la fermentation en milieu solide avec la PC comme inducteur et la bagasse de canne à sucre comme support. Globalement, cette étude a contribué à la découverte des nouvelles PLA et au criblage des PLAs à haut débit dans les extraits microbiens
Phospholipase A are lipolytic enzymes that hydrolyze selectively the ester bond of glycerophospholipid substrates at the sn-1 (PLA1) and sn-2 (PLA2) position, respectively, releasing free fatty acids and lysophospholipids. We identified new PLAs from fungi and actinomycetes isolated from Mexican environments. Firstly, we implemented an agar-plate method containing rhodamine 6G and phosphatidylcholine (PC) for the primary screening. Then, a colorimetric high-throughput screening assay for general PLA activity detection (cHTS-PLA) using PC substrate and cresol red was developed. According to 16S rRNA analysis, PLA producing strains were mostly belonging to Streptomyces and Aureobasidium genus. Production of PLAs was implemented by solid-state fermentation with PC as inductor and sugar-cane bagasse as support. Overall, this study has contributed to the discovery of new microbial PLAs and to pave the way toward the HTS of PLA activity in microbial extracts

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.

The cyclic pentapeptide c2 is a promising anti-cancer agent. The thesis investigates the mechanism of action of c2, focusing on its ability to prevent the interaction between hGIIA and vimentin. Computational studies using molecular dynamics and docking predicted that hGIIA would be stable as a monomer in physiological environments and provided possible binding conformations of c2 compound in hGIIA and vimentin. Using recombinant hGIIA and vimentin, assays were conducted to investigate the binding of hGIIA to vimentin and the role of c2 in inhibiting the interaction. Surface plasmon resonance and enzyme immunoassay studies showed evidence of hGIIA binding at the coil 2 domain of vimentin, and that c2 can inhibit this binding. Radiolabelled c2 displayed high affinity towards the coil 2 domain of vimentin relative to the coil 1 domain, suggesting that c2 prevents the interaction between hGIIA and vimentin possibly by binding to the hGIIA binding site on vimentin. From these results combined with the computational docking work, it is predicted that the most probable binding site of c2 on vimentin is located between Met347 and Asn357. In the advanced prostate cancer cell line PC-3, exogenously added hGIIA did not significantly increase the proliferation rate of the cells, and nor was this affected by vimentin silencing. The prostaglandin E2 level in PC-3 cells were reduced by the exogenously added hGIIA, and c2 had an opposing effect to hGIIA. Such results are a contrast to data that has come from early stage prostate cancer cells. The role of hGIIA potentially has changing roles depending on the stage and type of the cancer, and the mechanism of action of hGIIA in the prostate cancer cells has the potential to be highly complex. It was also demonstrated that c2 can bind to prostate cancer cells without the expression of hGIIA. It suggests the incorporation of c2 may be dependent on cell surface proteins such as vimentin to which it has affinity rather than hGIIA.

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.

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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
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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

Orientador: Marcos Hikari Toyama
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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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

Background: Platelet activity plays a role in the occurrence of diabetic angiopathy with an increase in mean platelet volume (MPV) as a marker of platelet activity. Platelet activity is influenced by phospholipase A2 type IIA (sPLA2 type IIA), which is a lipid-mediating enzyme that connects the pathogenesis of Diabetes Mellitus (DM) with complications of diabetic angiopathy. This study aimed to examine the relationship between levels of type IIA sPLA2 and MPV among type II DM patients. Subjects and Method: This was a cross-sectional study. A total of 63 patients with type II DM was selected for this study. The inclusion criteria for the study subjects were type 2 diabetes mellitus patients who did not experience an infectious disease, acute inflammation, trauma, surgery or malignancy, anemia, taking antiplatelet drugs, having abnormal platelet counts, and smoking. The dependent variable was levels of type IIA sPLA2. The independent variable was MPV. The data were obtained from the medical records of Prof. Dr. Soerojo Mental Hospital, Magelang. The data were analyzed using Spearman correlation test. Results: The study showed the median level of sPLA2 type IIA was 3841.50 ng / dL and the average MPV value was 7.36 fl. The results of the Spearman correlation analysis showed that there was no relationship between sPLA2 type IIA and MPV (p = 0.551), but there was a tendency for an increase in type IIA sPLA2 followed by an increase in MPV value (r = 0.077). There was a difference in the average MPV value in the subject group with DM ≤ 10 years and> 10 years (p = 0.009), and it was statistically significant. Conclusion: There is a tendency for an increase in type IIA sPLA2 followed by an increase in the MPV value among type II DM patients. Keywords: type II diabetes melitus, type IIA sPLA2 enzyme, mean platelet volume Correspondence: Woro Ayu Sekararum. Faculty of Medicine, Universitas Pembangunan Nasional ‘Veteran’ Jakarta. Jl, Rumah Sakit Fatmawati, Pondok Labu, South Jakarta, Indonesia. E-mail: woroayu.sekararum@gmail.com. Mobile: 0811975511 DOI: https://doi.org/10.26911/the7thicph.01.09