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.

Phosphatidylinositol is a a minor membrane component of eukaryotic cells, however, it plays a crucial role in cell signaling pathways as a precursor for a number of signaling molecules and second messengers. Among the most significant ones are phosphoinositides created by phosphorylation of the hydroxyl groups of phosphatidylinositol at positions 3,4, and 5 of the inositol ring. Despite its significance, the spatial and temporal distribution and dynamics of phosphatidylinositol remains unclear owing mainly to the lack of a specific optical probe (biosensor) to visualize phosphatidylinositol in living cells. Biosensor for inositol phospholipids are based on lipid-binding domains of their effector proteins with high enough affinity and specificity for a given phosphoinositide - but nosuch domain is known for PI. However, an enzyme - phosphatidylinositol-dependent phospholipase C - that specifically recognizes phosphatidylinositol is known. This enzyme catalyzes the hydrolysis of phosphatidylinositol into diacylglycerol and inositol 1-phosphate and unlike eukaryotic homologs does not act upon the phosphorylated forms of phosphatidylinositol. The main aim of this thesis was to solve the structures of several inactive mutant forms of phospholipase C from Bacillus cereus complexed to myo-inositol which...

Aspergillus fumigatus has long been a focus of research, as it is the cause of the majority of Aspergillus infections. A. fumigatus is widely distributed in the environment and mainly distributed in air as conidia and is the main source of lung infection. A. fumigatus airborne counts were determined monthly during two years from the outside air environment at the University of Manchester campus and compared to total fungal airborne counts. Total fungal airborne counts were strongly seasonally associated with peak counts occurring during the summer months reaching 1,100-1400 CFU m-3and were correlated positively with mean temperature (R2=0.697). In contrast, Aspergillus fumigatus counts were not seasonally associated and gave persistent low levels of between 3-20 CFU m-3and were not correlated with mean temperature. A random selection of Manchester environmental isolates collected over one year along with clinical patient isolates and environmental isolates from the air from Dublin were analysed for genetic diversity using two combined RAPD primers. RAPD analysis revealed that the Manchester environmental isolates represented a genetically diverse population while the clinical isolates were less diverse and formed three major clusters. The Dublin isolates were the least diverse, probably due to their isolation at a single time point. When the pathogenicity of clinical and Dublin isolates were compared with a random selection of Manchester isolates in a wax moth model, as a group, clinical isolates were significantly more pathogenic than environmental isolates. Moreover, when relative pathogenicity of individual isolates was compared, clinical isolates were the most pathogenic, Dublin isolates the least pathogenic and Manchester isolates showed a range of pathogenicities suggesting that selection for the most pathogenic isolates from the environment occurs during patient infection. When the expression of secreted phospholipases in vitro during wax moth larvae of a range of isolates displaying varying degrees of pathogenicity was compared, two phospholipase C genes, AfplcA and AfplcC were strongly correlated with pathogenicity. AfplcC was by far the most highly expressed, however a ΔAfplcC knockout strain did not show attenuated virulence compared to the wild type in wax moth larvae.

Les diacylglycerol kinases catalysent la phosphorylation du diacylglycérol en acide phosphatidique. Nous avons montré que la PLC spécifique des phosphoinositide (PI-PLC) et la diacylglycérol kinase (DGK) régulent négativement l'expression basale de la plupart des gènes DREB2 dans les cellules en suspension d'Arabidopsis thaliana. Les gènes DREB2 codent pour des facteurs de transcription qui se lient aux motifs DRE (Drought Responsive Elements). Ces éléments sont également liés par les facteurs DREB1. Alors que les facteurs DREB2 sont principalement impliqués dans les réponses à la sécheresse et au stress chaud, les DREB1 sont quant à eux induits en réponse au froid. Nous avons également pu montrer que l'inhibition par des agents pharmacologiques des activités PI-PLC ou DGK conduit à l'induction de l'expression basale des gènes DREB1. Cependant, l'induction est beaucoup moins marquée chez les gènes DREB1 que DREB2A, un membre de la famille DREB2. Cela indique que les gènes DREB1 et DREB2, ne sont pas soumis à la même régulation transcriptionnelle et que la signalisation lipidique pourrait en partie expliquer les différences dans la régulation des gènes DREB. Les DGK d'Arabidopsis sont codées par une famille multigénique de 7 gènes. Parmi ces gènes, on retrouve la DGK5 dont les le transcrit peut subir un épissage alternatif, ce qui aboutit à deux transcrits, dont l'un comporte une protéine avec un domaine putatif de liaison à la calmoduline. Le mutant knock-out dgk5.1 à une racine plus courte lorsqu'il est cultivé à 12°C comparé au sauvage. Ce phénotype racinaire est corrélé avec une zone méristématique et des cellules plus petites. La croissance des racines du mutant n'est n'est pas modifiée en présence de la plupart des hormones testées. Pourtant, elle est moins sensible à l'auxine exogène à 12°C par rapport au WT. Le mutant dgk5.1 génère moins de racines secondaires en présence d'auxine exogène que le WT. Le promoteur DR5 n'est pas activé dans le mutant à 12°C par l'IAA exogène dans la zone méristématique, alors qu'il est dans le WT. Nos résultats montrent que le mutant dgk5.1 est altéré dans sa réponse à l'auxine à 12°C, suggérant un rôle de perception/transduction de l’auxine dans les racines courtes. / Diacylglycerol kinases catalyse the phosphorylation of diacylglycerol into phosphatidic acid. We show that phosphoinositide dependent-phospholipase C (PI-PLC) and diacylglycerol kinase (DGK) in Arabidopsis thaliana suspension cells negatively regulated the basal expression of most DREB2 genes. DREB2 genes encode transcription factors that bind to Drought Responsive Elements (DRE). Those elements are also bound by DREB1 factors. While DREB2 factors are mostly involved in drought and heat responses, DREB1s are induced in the response to chilling. We show also that the pharmacological inhibition of PI-PLC or DGK leads to the basal induction of DREB1 genes. However, the induction is much less marked for the DREB1 genes than that of DREB2A, a member of the DREB2 family. This illustrates that DREB1 and DREB2 genes, while having the same targets, are not submitted to the same transcription regulation, and that lipid signalling might in part explain these differences in the regulation of the DREB genes. In Arabidopsis, DGKs are encoded by a multigenic family of 7 members. In this thesis, we focus on DGK5. The transcripts can have differential splicing, leading to two mature transcript, one of which leading to a protein with a putative calmodulin binding domain. A dgk5 knocked-out mutant is comparable to the WT, except for shorter root when grown at 12°C. This short root phenotype is correlated with to shorter meristematic zone and smaller cells. The short root phenotype is not altered in presence of most hormones. Yet, the root growth is less sensitive to exogenous auxin at 12°C compared to the WT. Accordingly the mutant produces less secondary roots in presence of exogenous IAA than the WT at 12°C. The DR5 promoter is not activated in the mutant at 12°C by exogenous IAA, in the meristematic zone, while it is in the WT. Our results show that the dgk5.1 mutant is impaired in auxin response at 12°C, suggesting a role of auxin perception /transduction in the short root phenotype.

Insulin resistance, a key risk factor for type 2 diabetes, can be defined as when cells fail to respond effectively to insulin. In striated muscle and fat, this manifests as impaired insulin-stimulated glucose uptake due to reduced plasma membrane insertion of the glucose transporter GLUT4. In cell culture models, insulin resistance induced by chronic exposure to insulin, endothelin-1 or glucosamine, is correlated with reduced immunoreactivity of the lipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in plasma membrane sheets. However, the reason for this decrease, and whether other factors that induce insulin resistance affect PtdIns(4,5)P2 levels, is unknown. Using L6 skeletal muscle myotubes and 3T3-L1 adipocytes, this project has investigated whether PtdIns(4,5)P2 levels are perturbed in insulin resistance induced by several factors, including exposure to insulin, oxidative stress, and treatment with tumour necrosis factor α, endothelin-1 or angiotensin II (Ang II).All these pre-treatments were found to abolish insulin-stimulated 3H 2-deoxy-glucose uptake, and significantly decrease PtdIns(4,5)P2 levels, measured in cell extracts by quantitative blotting using a PtdIns(4,5)P2-specific probe, developed from the PH domain of phospholipase C (PLC) δ. Importantly the ability of insulin to stimulate glucose uptake can be restored by replenishing PtdIns(4,5)P2 in L6 myotubes treated with insulin and Ang II. PtdIns(4,5)P2 levels are regulated by three families of proteins; PIP kinases, which synthesise it, phosphatases, which remove phosphate groups from the inositol headgroup, and PLCs, which hydrolyse it. Membrane preparations from Ang II- and insulin-induced insulin resistant L6 myotubes showed no differences in PtdIns(4,5)P2 production or dephosphorylation. However a significant increase in PLC activity was detected in membranes from insulin resistant cells and membrane localisation of PLCβ family members was increased in insulin resistant cells. Furthermore, studies using PLC inhibitors show a restoration of PtdIns(4,5)P2 levels in insulin resistant cells, leading to partial reversal of insulin resistance.This study therefore shows a causal link between decreased PtdIns(4,5)P2 levels and insulin resistance in L6 myotubes, and that PLCs are the reason for the PtdIns(4,5)P2 decrease in Ang II- and insulin-induced insulin resistance. PLCs, or their activation pathways, may thus be a novel target for combating insulin resistance, and preventing type 2 diabetes.

Técnicas espectroscópicas vibracionais Raman e infravermelho foram utilizadas no estudo conformacional de algumas proteínas de interesse biológico, no que se refere a aspectos estruturais de seus esqueletos polipeptídicos e microambientes de cadeias laterais de certos resíduos de aminoácidos. O trabalho foi dividido em dois grupos, de acordo com os sistemas em estudo: a) Toxina γ do veneno de escorpião brasileiro Tityus serrulatus. A análise vibracional revelou que o esqueleto polipeptídico dessa proteína consiste de diferentes estruturas secundárias, com predominância de folhas β, seguida por estruturas do tipo não regular e α-hélice, com alguma evidência de dobras β. Conformação gauche-gauche-gauche foi detectada para os segmentos CCSSCC das quatro pontes dissulfeto. A intensidade do dubleto Raman da Tyr a 853 e 828 cm-1 indicou que 4 dentre 5 resíduos de Tyr encontram-se expostos na superficie molecular. Também há indicações de que os 3 resíduos de Trp apresentem localização externa. Sob o ponto de vista qualitativo, as características conformacionais da toxina no estado sólido amorfo e em solução são virtualmente as mesmas. b) Fosfolipases A2 de pâncreas de porco e de Crotalus durissus terrificus. Mudanças confarmacionais foram detectadas para as moléculas de fosfolipase como consequência de diferentes condições experimentais tais como mudança de estado físico, presença de certas espécies iônicas e interação com um análogo de substrato e com o próprio substrato. Características conformacionais discrepantes foram observadas para a forma sólida amorfa e forma cristalina da fosfolipase pancreática. Transições conformacionais foram detectadas para a transformação zimogênio → fosfolipase A2 e diferentes conteúdos estruturais foram calculados para a forma tóxica e atóxica dessa enzima. Todas essas mudanças conformacionais envolveram basicamente a arquitetura do esqueleto polipeptídico, não afetando a conformação das cadeias laterais dos resíduos de amino ácidos. As pontes dissulfeto apresentaram consistentemente uma conformação ggg a qual não foi perturbada por nenhuma das condições experimentais empregadas. A ocorrência externa de resíduos de triptofano constituiu uma característica comum para os sistemas ensaiados, assim como a localização predominante de residuos de tirosina em microambientes hidrofílicos, provavelmente na superficie molecular. / Raman and infrared spectroscopies were used to investigate conformational features of some proteins of biological interest, in what concerns structural aspects of their polypeptide backbones and microenvironments of certains amino acid residue side-chains. The work has been divided in two groups as related to the systems studied: a) Toxin γ from the venom of Brazilian scorpion Tityus serrulatus. The vibrational analysis has revealed that the protein polypeptide backbone consists of different secondary structures, with predominance of β-sheet, followed by unordered structure and α-helix, with some evidence of β-turns. A gauche-gauche-gauche (ggg) conformation for the CCSSCC fragments of the four dissulfide bridges has been detected. The intensity of the Tyr Raman doublet at 853 and 828 cm-1 indicated that 4 out of the 5 Tyr residues are exposed at the molecular surface. External localization of the 3 Trp residues has also been indicated. Under a qualitative point of view, conformational features of the toxin the amorphous solid state and in solution were virtually the same. B) Crotalus durissus terrificus and porcine pancreatic phospholipases A2. Conformational changes were detected for the phospholipases molecules as a consequence of different conditions such as change of physical state, presence of certain ionic species and interaction with a model substrate analog and with the substrate itself. Amorphous and crystalline solid pancreatic phospholipases presented discrepant conformational features. Conformational transitions were detected for the pancreatic zymogen → phospholipase A2 transformation and different secondary structures contents were observed for the toxic and the non toxic phospholipase melecules. All those structural changes heve been shown to involve primarily the architecture of the polypeptide backbone rather than the conformation of amino acid residue side-chains. Disulfide bridges have shown consistently a ggg conformation which has not been disturbed by any of the experimental conditions employed. The external occurrence of tryptophan residues has been a common feature for the systems assayed, as well as the predominant localization of tyrosine residues in hydrophylic environments, probably at the molecular surface.

A crotoxina é a toxina mais abundante e ativa do veneno da cascavel brasileira Crotalus durissus terrificus. É composta por duas sub-unidades ligadas não covalentemente. A sub-unidade ácida é enzimaticamente inativa e não possui toxicidade, é também conhecida como crotapotina e potencializa a ação da fosfolipase A2 (sub-unidade básica). Alguns autores atribuem à crotoxina um efeito citotóxico e sugerem seu uso como um agente terapêutico contra tumores. Neste trabalho, foi investigada a citotoxidade da crotoxina e suas subunidades em células de melanoma murino e fibroblastos. Os ensaios de indicam que a crotoxina é tóxica para as células de melanoma promovendo a morte até em concentrações mais baixas enquanto os fibroblastos foram afetados somente em concentrações mais altas. Ensaios com as subunidades mostraram que a fosfolipase A2 foi mais tóxica para as células de melanoma que para os fibroblastos. / Crotoxin is the most abundant and active toxin from the venom of the Brazilian rattlesnake Crotalus durissus terrificus. It is composed of two subunits non covalently linked. One acidic with no enzymatic or toxic activity, known as crotapotin, which enhances the phospholipase A2 (basic subunit) action. Some authors have also attributed to this toxin a direct cytotoxic effect, and have suggested its use as a therapeutical agent against tumoral cells. In the present work, we investigated the cytotoxic effect of crotoxin and its subunitis on murine melanoma cells and fibroblasts. Our results indicate that crotoxin is highly toxic to the melanoma cells inducing cell death even at the lowest concentration while the fibroblast were only affected with the higher concentrations. The subunits assays showed that the phospholipase A2 had a higher toxicity on melanoma cells than on fibroblasts.

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 (γPLI) 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.

<p>Phospholipase Cs (PLCs) are a family of enzymes that hydrolyze membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to generate inositol triphosphate (IP3) and diacylglycerol (DAG). These second messengers activate a variety of intracellular responses, including inflammation, vascular smooth muscle contraction, and cardiac hypertrophy. While much is known about how Gaq-mediated activation of PLCb occurs, the same cannot be said for Gbg-mediated activation. Residues within the PLCb-Gbg binding interface were previously identified in interior regions of the protein, suggesting the PH domain must undergo a conformational change to allow for Gbg-mediated activation. However, the role of PH domain conformational dynamics in Gbg-mediated activation of PLCb has yet to be determined. In this work, I discuss efforts to characterize conformational dynamics of the PLCb PH domain and its role in interactions of the enzyme with liposomes and Gbg. First, I generated a disulfide crosslink between the PH domain and EF hands1/2 of PLCb3, purified under oxidizing or reducing conditions, and conducted biochemical and structural tests to determine any differences in structure and/or function of the protein as compared to wild-type. Results of these studies provided the first direct structural evidence of PLCb PH domain dynamics in solution. Then, I discuss the rationale behind the generation of a surface cysteine-less PLCb for use in solvatochromic fluorescence assays in the presence and absence of liposomes and Gbg. Initial results of these studies suggest the PLCb PH domain favors a buried conformation alone and in the presence of Gbg or liposomes, and likely exists at an equilibrium between open and closed states.</p>

Candida albicans is an increasingly important pulmonary fungal pathogen. Resident alveolar macrophages are important in host defense against opportunistic fungal infections. Activation of Group IVA cytosolic phospholipase A2α (cPLA2α) in macrophages initiates arachidonic acid (AA) release for production of eicosanoids, which regulate inflammation and immune responses. We investigated the ability of C. albicans to activate cPLA2α in unprimed alveolar macrophages and after priming with granulocyte macrophage colony-stimulating factor (GM-CSF), which regulates alveolar macrophage maturation. AA was released within minutes by GM-CSF-primed but not unprimed alveolar macrophages in response to C. albicans, and was blocked by soluble glucan phosphate (S-GP). The expression of the β-glucan receptor dectin-1 was increased in GM-CSF-primed macrophages, and AA release from GM-CSF-primed dectin-1-/- alveolar macrophages was reduced to basal levels. The enhanced activation of extracellular signal-regulated kinases and phosphorylation of cPLA2α on Ser-505 that occurred in GM-CSF-primed macrophages were reduced by MEK1 and Syk inhibitors, which also suppressed AA release. At later times after C. albicans infection (6 h), unprimed and GM-CSF-primed macrophages released similar levels of AA. The expression of cyclooxygenase 2 and prostanoid production at 6 hours was higher in GM-CSF-primed macrophages, but the responses were not dependent on dectin-1. However, dectin-1 contributed to the C. albicans-stimulated increase in TNF-α production that occurred in GM-CSF-primed macrophages. The results demonstrate that dectin-1 mediates the acute activation of cPLA 2α in GM-CSF-primed alveolar macrophages, but not in the more delayed phase of AA release and GM-CSF-dependent prostanoid production.