As fosfolipases A2 secretadas (sFLA2) de veneno de serpente apresentam homologia estrutural e funcional com as sFLA2s do GIIA de mamíferos, cujos níveis estão elevados em doenças inflamatórias, como a aterosclerose. Nesta doença, as células de músculo liso vascular (CMLVs) acumulam corpúsculos lipídicos (CLs) e se diferenciam em células espumosas. Porém, o papel das sFLA2s neste fenômeno não é conhecido. Neste estudo foram avaliados os efeitos das FLA2 MT-III, cataliticamente ativa, e da BthTx-II, sem atividade catalítica, em CMLVs, com ênfase na formação de CLs e a participação de fatores da homeostasia lipídica. Os resultados obtidos demonstraram que a MT-III e a BthTx-II induziram a formação de CMLVs espumosas. Para tanto, estas enzimas recrutaram diferentes fatores envolvidos na síntese e acúmulo de lipídios. Nesta condição, os CLs constituem um local de síntese de prostaglandinas. Ainda, a MT-III induziu a diferenciação de CMLVs para fenótipo e função de macrófagos. A atividade catalítica não é relevante para a formação de CLs induzida por FLA2s. / Bothrops snake venom secreted phospholipases A2 (sPLA2s) share structural and functional features with mammalian GIIA sPLA2s, which are highly expressed during inflammatory diseases, such as atherosclerosis. In this disease, vascular smooth muscle cells (VSMCs) are loaded with lipid droplets (LDs) differentiating into foam cells. However, the role of these enzymes in this process is still unknown. In this study the effects of snake venom PLA2s MT-III with catalytic activity and BthTx-II, devoid of catalytic activity in VSMCs, with focus on LDs formation and mechanisms involved were investigated. Results here obtained show that both MT-III and BthTx-II induce formation of foam VSMCs and recruit distinct factors of synthesis and storage of lipids in these cells. In this condition, LDs constitute sites for synthesis of prostaglandins. Moreover, MT-III showed the ability to modulate VSMCs functions, leading them to a phenotipic switch to macrophage-like cells. In addition, the catalytic activity is not relevant to sPLA2-induced LDs formation.

As fosfolipases A2 (EC 3.1.1.4) catalisam a hidrólise das ligações ácido-éster na posição sn-2 de glicerofosfolipídios liberando, como produto da catálise, ácidos graxos e lisofosfolipídios. Membros da sub-família de fosfolipases A2-Lisina49 (PLA2-Lys49), isolados de venenos de serpentes Viperidae mostram uma substituição do resíduo de aspartato na posição 49 por uma lisina, com a eliminação concomitante da atividade hidrolítica contra fosfolipídeos. Apesar da ausência de atividade catalítica, as PLA2-Lys49 apresentam propriedades farmacológicas variadas incluindo miotoxicidade, e danifica membranas artificiais por um mecanismo Ca2+-independente, que não envolve hidrólise de fosfolipídeos. As PLA2-Lys49 formam homodímeros em solução, e estudos cristalográficos e espectroscópicos de Bothropstoxina-I, uma PLA2-Lys49 do veneno de Bothrops jararacussu, revelaram que a transição na estrutura quaternária do dímero provoca a mudança de posição da região C-terminal, apoiando a sugestão do envolvimento desta região no modelo proposto de danificação da membrana Ca2+-independente. Um papel para a região Cterminal das PLA2-Lys49 também foi sugerido na atividade bactericida observada para esta proteína, e o presente estudo investiga uma possível correlação entre a atividade de danificação de membranas Ca2+-independente e o efeito bactericida. O efeito bactericida de BthTx-I e mutantes da proteína na região C-terminal foi avaliado contra bactéria Escherichia coli (K12). A BthTx-I nativa e recombinante tiposelvagem apresentaram alta atividade bactericida com uma concentração de 5 mg/mL, porém as mutantes Y117W, Y119W, K122A e F125W reduziram significativamente este efeito, mostrando uma correlação entre os determinantes estruturais das atividades bactericida e de danificação em membranas artificiais. Na tentativa de correlacionar o mecanismo de danificação de membranas artificiais com a atividade bactericida de BthTx-I contra linhagem E.coli (K12), um estudo por partição de sondas fluorescentes em compartimentos celulares específicos foi realizado. A sonda hidrofóbica N-fenil-N-naftilamina (NPN) foi utilizada para avaliar a integridade da membrana externa da bactéria e a sonda Sytox Green (SG) para avaliar a integridade da membrana citoplasmática bacteriana. A cinética de permeabilização da membrana externa é rápida e não foi influenciada por mutagênese da região C-terminal da proteína. Entretanto, a cinética de permeabilização da membrana plasmática mostrou-se lenta, com um efeito máximo de 2 horas de ação e identificou os mesmos determinantes estruturais como os identificados para a atividade bactericida de BthTx-I. Resultados obtidos pelas técnicas de citometria de fluxo e microscopia eletrônica de transmissão auxiliaram os dados evidenciando a importância da região C-terminal de BthTx-I na atividade bactericida contra linhagem E.coli. / Phospholipases A2 (PLA2 - EC 3.1.1.4) catalyze the hydrolysis of acid ester bonds at the sn-2 position of glycerophospholipids liberating fatty acids and lysophospholipids as catalysis products. Lysine 49 phospholipase A2 (Lys49-PLA2) are isolated from the venom of viperid snakes, and in these proteins, the aspartic acid at position 49 is replaced by a lysine, resulting in the elimination of hydrolytic activity against phospholipid substrates. Despite the absence of catalytic activity, these Lys49-PLA2s present various pharmacological properties and furthermore damage artificial membranes by a Ca2+-independent mechanism. Lys49- PLA2s form homodimers in solution, and crystallographic and spectroscopic studies of the bothropstoxin-I (BthTx-I), a Lys49-PLA2 isolated from venom of Bothrops jararacussu, reveal that a quaternary structure transition in the homodimer results in a change in the position of the C-terminal loop of the protein, suggesting the involvement of this region in the Ca2+- independent membrane damaging activity. A role for the C-terminal region of Lys49-PLA2 has also been suggested for the bactericidal activity of theses proteins, and using BthTx-I one as a model system, the present study investigates the possible correlation and between the Ca2+-independent membrane damaging and the bactericidal activities. The bactericidal effect of native BthTx-I and site-directed mutants of the C-terminal loop was evaluated using the Gram negative bacteria E. coli strain K12. Both the native and wild type recombinant BthTx-I presented a high bactericidal activity at a concentration of 5 mg/mL, whereas the mutants Y117W, Y119W, K122A and F125W showed significantly reduced the bactericidal effects, showing a correlation between the structural determinants of the bactericidal and membrane damaging activities. The fluorescent probe NPN was used to evaluate the integrity of the external membrane of the bacterial cells after exposure to the BthTx-I and mutants. The permeabilization of the external membrane is complete within 2 minutes, and neither the kinetics nor the extent of membrane damage was influenced by mutagenesis in the C-terminal region. The fluorescent probe Sytox Green (SG) was used to evaluate the integrity of the bacterial plasma membrane, an event which showed a significantly slower kinetic, with a maximum effect observed after two hours exposure to the BthTx-I. Furthermore, the extent of the membrane damage is influenced by mutagenesis in the C-terminal loop, and the structural determinants for the bactericidal activity of BthTx-I are the same as those that determine the permeabilization of the bacterial plasma membrane. Evidence obtained using flow cytometry and transmission electron microscopy support of the suggestion that the C-terminal region of BthTx-I is an important structural determinant of the plasma membrane damaging bactericidal activities.

A cristalografia de raios-X e um método de singular importância para a determinação da estrutura de macromoléculas. A importância em resolver estruturas de proteínas continua a crescer em campos variando desde a bioquímica e biofísica básicas ate o desenvolvimento farmacêutico e biotecnologia. o presente trabalho esta relacionado com os estudos cristalográficos de três diferentes moléculas biológicas. Calgranulina C de granulócitos porcinos, uma proteína que liga cálcio, supostamente envolvida em processos celulares regulados, foi cristalizada com parâmetros de rede a=b=54.35 e c=141.32&#197, grupo espacial P3121 ou seu enantiomorfo P3221. Várias tentativas foram feitas no sentido de se determinar a estrutura por substituição molecular, mas a alta flexibilidade entre os motivos \"EF-hands\" quando da ligação ao íon cálcio podem ser responsáveis pelo insucesso dos resultados. Tripanotiona redutase de T. cruzi e um excelente alvo para modelagem de inibidores e potenciais drogas contra a doença de Chagas. O complexo mutante TR + GSPD foi cristalizado com parâmetros de rede a=b=92.7 e c=156.2&#197, grupo espacial P43. Um conjunto preliminar de fases foi obtido por refinamento de corpo rígido contra as coordenadas da TR nativa. O modelo foi refinado a 2.4&#197 de resolução, Rfactor=19.8%. Fosfolipase A2 extraída do veneno da serpente Bothrops moojeni foi cristalizada com parâmetros de rede a=63.1, b=90.5 e c=40.2&#197 e &#946=125.1&#176, grupo espacial C2. A estrutura foi resolvida por substituição molecular usando o dímero da fosfolipase A2 de Crotalus atrox como modelo. A analise de sua seqüência de aminoácidos e necessária para posteriores ciclos de refinamento. / X-ray crystallography is a essential method for determination of the structure of macromolecules. The importance of solving protein structures continues to grow in fields ranging from basic biochemistry and biophysics to pharmaceutical development and biotechnology. The current work is concerned to the crystallographic studies of three different biological molecules. Calgranulin C from pig granulocytes, a calcium binding protein though to be involved in regulation of cell process, was crystallized with cell parameters a=b=54.35 and c=141.32&#197, space group P3121 or its enantiomorph P3221. Several attempts were made in order to solve the structure, but the high flexibility between its EF-hands motives while binding the ion calcium may be responsible for the lack of success in the results. Trypanothione reductase (TR) from T. cruzi is a target enzyme for modeling an inhibitor for Chagas´ disease. The C58S TR + GSPD mutant complex was crystallized with a=b=92.7 and c=156.2&#197, space group P43. A preliminary set of phases was obtained by rigid body refinement against the coordinates for the native TR. The model was refined to 2.4&#197 resolution, Rfactor=19.8%. Phospholipase A2 extracted from the venon of the snake Bothrops moojen; was crystallized with cell parameters a=63.1, b=90.5, c=40.2&#197 and &#946=125.1&#176, space group C2. The structure was solved by molecular replacement techniques using the dimer of phospholipase A2 from Crotalus atrox as a model. The aminoacid sequence analysis is needed for further refinement cycles.

Abstract not available

共著者あり。共著者名:Munakata Mitsuru, Sato Atsuko, Amishima Masaru, Homma Yukihiko, Kawakami Yoshikazu. / Hokkaido University (北海道大学) / 博士 / 医学

<p>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.</p><p>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.</p><p>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.</p>

The goal of the present study is to elucidate the modulatory effect of lipid compositions on phospholipase A2 (PLA2) activity. Sphingomyelin (SM) incorporation inhibited catalytic activity and membrane-damaging activity of native and mutated PLA2 toward egg yolk phosphatidylcholine (EYPC) vesicles. The inhibitory effects were through the reduction of membrane fluidity and modulation of the mode of membrane binding of PLA2 at water/lipid interface. The modulated effect of SM depended on inherent structural elements of PLA2. Moreover, cholesterol (Chol) incorporation into EYPC/egg yolk sphingomyelin (EYSM) vesicles relieved the inhibitory effect of sphingomyelin on PLA2 activity via lipid domain formation by SM and Chol. The effects on the interactive mode of PLA2 with phospholipids induced by the physical state changes of membrane bilayers abolished the inhibition of SM on catalytic activity and membrane-damaging activity of PLA2. Additionally, quercetin incorporation increased PLA2 activity and membrane-damaging activity toward EYPC/SM vesicles via its raft-making effect. Quercetin incorporation reduced PLA2 activity and membrane-damaging activity toward EYPC/SM/Chol vesicles via its raft-breaking effect. Membrane-inserted quercetin affected on membrane structure and membrane-bound mode of PLA2 to modulate PLA2 interfacial activity and membrane-damaging activity. Finally, studies on the effects of phosphatidylserine (PS) content on the sensitivity of lipid vesicles mimicking inner and outer plasma membrane toward PLA2 activity revealed that the membrane-binding mode adopted by PLA2 depended on the lipid composition. The effects of PS content on the extent of lipid domain formation and the conformation of PLA2 adopted at water-lipid interface modulate PLA2 catalytic activity. Collectively, these results indicate that lipid composition modulates PLA2 activity via its effects on membrane structure and membrane-bound mode of PLA2

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / School of Physical Education, Sport, and Exercise Science

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / School of Physical Education, Sport, and Exercise Science

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / School of Physical Education, Sport, and Exercise Science