Estudos têm demonstrado que o microambiente tumoral é rico em ATP e adenosina, sugerindo o envolvimento da sinalização purinérgica no desenvolvimento e/ou manutenção do câncer. Ainda, o receptor purinérgico P2X7, conhecido pelo seu papel na indução de apoptose, encontra-se reduzido em alguns tecidos tumorais em comparação aos tecidos saudáveis, indicando que a sua redução possa ser um mecanismo de resistência celular à apoptose. Dessa forma, compreender o papel da sinalização purinérgica no contexto do câncer se torna indispensável e permite que novas abordagens terapêuticas sejam implementadas. Nesse trabalho, avaliamos a função dos nucleotídeos e nucleosídeos da adenina, bem como do receptor P2X7 na indução da morte celular em células de câncer cervical. Também verificamos o efeito do heat shock na potencialização da atividade do receptor P2X7 frente à curta exposição ao ATP em células de câncer de cólon. De acordo com os nossos resultados, o efeito citotóxico do ATP extracelular nas linhagens de câncer cervical é mediado principalmente pela ação do seu metabólito adenosina, que ao entrar no interior das células, promove o aumento dos níveis intracelulares de AMP, ativação de AMPK, aumento da p53 e indução de autofagia. O papel do receptor P2X7 nesse contexto parece ser apenas coadjuvante, visto que o seu bloqueio ou silenciamento impediu em apenas 20% a morte celular. Além disso, utilizando células de câncer de cólon, nós demonstramos que o heat shock aumenta a funcionalidade do receptor P2X7, independente da interação com heat shock proteins ou canais do tipo conexina/panexina, potencializando o efeito citotóxico do ATP. Esse efeito parece estar relacionado à mudanças na composição e arquitetura da membrana celular, visto que o uso do agente fluidizador de membrana benzil álcool foi capaz de mimetizar o efeito do heat shock na potencialização do receptor P2X7 a 37ºC. Este estudo fornece evidências adicionais sobre o papel da sinalização purinérgica no contexto da biologia celular tumoral e abre novas perspectivas para o uso dos nucleotídeos de adenina associados a hipertermia como agentes adjuvantes na terapia do câncer. / The tumor microenvironment is rich in ATP and adenosine, suggesting an involvement for purinergic signaling in cancer development and surveillance. The P2X7 receptor, among the P2 purinergic receptors, is broadly recognized as the “death receptor”, because it promotes cell apoptosis when exposed to high levels of extracellular ATP. Researches have been shown that P2X7 protein levels are decreased at the tumor site in comparison to adjacent healthy tissue, suggesting a mechanism of tumor escape to cell death. Thus, understanding purinergic signaling in a cancer context becomes urgent and opens a new field for therapeutic strategies. Here, we evaluated adenine nucleotides and nucleosides cytotoxicity, as well as P2X7 role in cell death induction using cervical cancer cell lines. Indeed, we investigated heat shock effect on P2X7 functionality through exposing colon cancer cell shortly to ATP at 40ºC. According to our data, adenosine uptake formed from ATP metabolism is the main responsible for the extracellular ATP cytotoxicity in cervical cancer cells. While inside of the cell, adenosine is converted to AMP, leading to AMPK activation, p53 increase and autophagy induction. ATP induced cell death per se through P2X7 in this context seems to be less important, since P2X7 blockage or knocking down reduced only 20% of cell death. In colon cancer cells, we found that heat shock stress was able to increase P2X7 pore formation independently of heat shock protein interaction or native pore-forming transporters association (e.g pannexin-or connexin-type channels), thus leading to an increase ATP cytotoxicity. The mechanism enrolled in this process seems to be related to changes in the lipid composition and architecture of membrane, as the membrane fluidizer benzyl alcohol could reproduce heat stress effect in potentiating P2X7 activation at 37ºC. In conclusion, our work provides further evidence for a purinergic signaling role in the cancer biology context and opens new perspectives for the utility of purine-based drugs associated to hypertermia as adjunctive agents in cancer therapy.

Binding of inorganic phosphate (Pi) in ATP synthase catalytic sites is a crucial step for the synthesis of adenosine-5'-triphosphate (ATP). ATP is the fundamental means of cellular energy in almost every organism, and in order to gain insight into the regulation of ATP catalysis, critical amino acid residues responsible for binding Pi must be identified. Here, we investigate the role of highly conserved α-subunit VISIT-DG sequence residues αSer-347, αGly-351, and αThr-349 in Pi binding. Mutations αS347A/Q, αG351Q, αT349A/D/R, βR182A, and αT349R/βR182A were generated via site directed mutagenesis. Results from biochemical assays showed that αSer-347 is required for transition state stabilization and Pi binding whereas αGly-351 is only indirectly involved in Pi binding and most likely maintains structural integrity of the catalytic site. Results from preliminary experiments on αThr-349 mutants suggest that the residue may be involved in Pi binding; however, further investigation is required to fully test this hypothesis.

The metabolic pathways required for adenosine triphosphate (ATP) production within the cell are well understood, however recent publications suggest that metabolic pathways are closely linked to immune cell activation and inflammatory diseases. There has been little examination of the metabolic pathways that modulate mast cell activation and the feedback regulator lactic acid. Here we examine metabolic pathways and regulation within mast cells in the context of lipopolysaccharide (LPS) and interleukin (IL-33) activation, for which there has been little to no reported studies. First, we examine the effects of lactic acid, previously considered only a by-product of glycolysis and now understood to act as a negative feedback regulator of inflammation in the context of LPS activation and sepsis. Lactic acid is elevated in septic patients and associated with mortality, potentially due to suppressive effects on LPS signaling and contribution to late phase immunosuppression. By attenuating glycolysis and reducing ATP availability for signaling and cytokine transcription, lactic acid impairs the function of immune cells to fight the initial or subsequent infections. We support this with in vitro and in vivo data. Additionally, our lab has published that lactic acid can suppress IL-33 activation, potentially by metabolic modulation as with LPS activation; however there has been no study of the metabolic requirements for IL-33 activation. We report here that glycolysis is required for ATP and reactive oxygen species (ROS) production to augment signaling and cytokine production downstream of the IL-33 receptor. Together, these studies examine the contribution of metabolism to mast cell activation and may provide potential targets for treatments of diseases that involve LPS- or IL-33-dependent mast cell activation.

Lactococci are widely used in the cheese industry as a starter culture. Starter cultures face carbohydrate starvation due to the absence of a fermentable carbohydrate in the cheese curd after pressing. Starvation leads to a decreased ability to synthesize ATP, generate a proton motive force, and accumulate nutrients necessary to maintain viability. The aim of this work was to investigate the culturability of lactococci grown with and without lactose in a chemically defined medium, and to define the metabolic changes that occur during carbohydrate starvation. Lactose metabolism provided energy for logarithmic phase growth and greater cell density in L. lactis ssp. lactis ML3 and L. lactis ssp. cremoris S2. However, the rate of lactose metabolism was strain dependent in that L. lactis ssp. lactis 11454 did not metabolize lactose as rapidly as did ML3 and S2. In the absence of lactose the cells became nonculturable on agar. In addition to becoming nonculturable, the aminopeptidase and lipase/ esterase activity became nonmeasurable after 21 d, and cellular metabolism was altered because of carbohydrate starvation. Nevertheless, the cells remained viable for up to 42 d in spent media as measured by fluorescent viability stains and intracellular ATP content. Fluorescent viability staining demonstrated that the cells maintained an intact cell membrane to contain their DNA, as well as to contain enzymes and ATP necessary to maintain viability and metabolic activity. With the addition of arginine to the basal medium, the survival time, cell number, and ATP concentration increased. Amino acids, including arginine, provided energy after carbohydrate exhaustion. At the onset of lactose exhaustion, the extracellular concentrations of arginine, glycine/valine, glutamate, and glutamine decreased in the media when energy was present for their transport. There was a significant increase in serine and methionine concentrations in the spent media over the same time period. These data indicated lactococci remained viable and metabolically active, but were nonculturable in response to carbohydrate starvation. Additionally, amino acids are in a dynamic state during carbohydrate starvation, and utilization of amino acids, such as arginine and serine, could facilitate lactococcal cells in maintaining viability in harsh environments such as ripening cheese.

Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X7 receptor. These responses include opening of a cation selective channel/pore which allows entry of the fluorescent dye, ethidium+ and activation of a membrane metalloprotease which sheds the adhesion molecule L-selectin. In this thesis, the surface expression of P2X7 receptors was measured in normal leucocytes, platelets and B-CLL lymphocytes and compared with their functional responses. Monocytes showed 4-5 fold greater expression of P2X7 than B-, T- and NK- lymphocytes, while P2X7 expression on neutrophils and platelets was weak. All cell types demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed surface P2X7 at about the same level as for B-lymphocytes from normal subjects. P2X7 function, measured by ATP-induced uptake of ethidium, correlated closely with surface expression of this receptor in normal and B-CLL lymphocytes and monocytes. However, the ATP-induced uptake of ethidium into the malignant B-lymphocytes in 3 patients was low or absent. The lack of P2X7 function in these B-lymphocytes was confirmed by the failure of ATP to induce Ba2+ uptake into their lymphocytes. This lack of function of the P2X7 receptor resulted in a failure of ATP-induced shedding of L-selectin, an adhesion molecule which directs the recirculation of lymphocytes from blood into the lymph node. To study a possible genetic basis of non-functional P2X7 receptor, we sequenced DNA coding for the carboxyl terminal tail of P2X7. In 33 of 130 normal subjects a heterozygous nucleotide substitution (1513A--C) was found while 3 subject carried the homozygous substitution which codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X7 on lymphocytes was not affected by this 496Glu--Ala polymorphism demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the 496Glu--Ala homozygote subject expressed non-functional receptor while heterozygotes showed P2X7 function which was half that of wild type P2X7. Results of transfection experiments showed the mutant P2X7 receptor was non-functional when expressed at low receptor density but regained function at a high receptor density. This density-dependence of mutant P2X7 function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma which upregulated mutant P2X7 and partially restored its function. P2X7-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X7 compared with wild type. The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X7 receptor. Apart from the 496Glu--Ala polymorphism, three other single nucleotide polymorphisms, 155His--Tyr, 348Ala--Thr and 568Ile--Asn were also found in the P2X7 receptor. The site directed mutant cDNA were generated for all 3 polymorphisms and transfected into HEK293 cells to study the impact of these polymorphisms on P2X7 function. Results suggested that Ile568 is important for P2X7 protein trafficking to cell surface. Further study of these two loss-of-function polymorphisms (496Glu--Ala and 568Ile--Asn) may help better understanding of the functional domains in the P2X7 receptor and its role in CLL, lymphoma and infectious diseases. Conclusions: 1.P2X7 receptor is expressed in human leukocytes, including lymphocytes, natural killer cells as well as monocytes, on both surface and intracellular locations. 2.Both the expression and function of P2X7 are highly variable between in human individuals. Non-functional P2X7 receptors are found in some subjects, including both normal subjects and CLL patients, and are often associated with defects in ATP-induced cytotoxicity and L-selectin shedding. 3.Two single nucleotide polymorphisms (SNPs), 496Glu--Ala and 568Ile--Asn, are found at low frequency in the human population and lead to the loss-of-function of P2X7. Both permeabllity function and the downstream effects mediated by P2X7 are affected by these two SNPs. The mechanisms for the loss-of-function differs between the two polymorphisms.

Master of Science / In vivo many forms of glia utilise both intercellular and extracellular pathways in the form of IP3 permeable gap junctions and cytoplasmic ATP diffusion to produce calcium waves. We introduce a model of ATP and Ca2+ waves in clusters of glial cells in which both pathways are included. Through demonstrations of its capacity to replicate the results of existing theoretical models of individual pathways and to simulate experimental observations of retinal glia the validity of the model is confirmed. Characteristics of the waves resulting from the inclusion of both pathways are identified and described.

Thesis (M.D.) -- University of Texas Southwestern Medical Center at Dallas, 2007. / Vita. Bibliography: pp.28-30

Le système de réparation des mésappariements de l'ADN (MMR) est impliqué dans la cytotoxicité de l'agent anticancéreux cisplatine en activant une voie apoptotique. La déficience de ce système de réparation est reliée in vivo à une chimiorésistance des cellules cancéreuses au cisplatine. Afin de définir le lien entre la cytotoxicité du cisplatine et le fonctionnement du système MMR, nous avons étudié l'interaction de la protéine MutS du système MMR bactérien avec un composé de lésion du cisplatine (formé lorsqu'une base non complémentaire est incorporée en face de l'une des deux guanines platinées appartenant à l'adduit intrabrin d(GpG)). Mon travail a porté essentiellement sur i) une étude des propriétés biochimiques ATP-dépendantes de MutS en présence d'un composé de lésion du cisplatineii) une étude d'interaction entre plusieurs composés de lésion du cisplatine et la protéine HMGB1 qui est bien connue comme pouvant inhiber l'accessibilité des lésions majoritaires du cisplatine à des protéines de réparation. Notre étude a été réalisée par des techniques de biologie moléculaire, de biochimie et de spectroscopie (résonance plasmonique de surface). Nous montrons qu'un composé de lésion du cisplatine module les propriétés ATP-dépendantes de MutS, un résultat inattendu étant qu'il inhibe le relargage de MutS de l'ADN. Un composé de lésion pourrait donc jouer un rôle dans la signalisation MMR-dépendante en modulant les stades précoces de l'initiation de la réparation ce qui est en accord avec le modèle dit « de signalisation directe MMRdépendante».

Le canal KATP résulte de l'assemblage d'un canal potassique inhibé par l'ATP intracellulaire (Kir6.2) et d'un transporteur<br />ABC, le récepteur des sulfonylurées (SUR) de la famille MRP/ABCC. SUR a un rôle régulateur essentiel : il confère au<br />canal une sensibilité accrue à l'inhibition par l'ATP, provoque son activation lorsque l'ADP augmente, et est la cible des<br />activateurs et bloqueurs pharmacologiques du canal.<br />Nous nous sommes intéressés à divers aspects structure/fonction de SUR en tant que modèle de transporteur ABC<br />eucaryote. Son couplage naturel à un canal ionique en facilite grandement l'étude grâce à la technique<br />électrophysiologique du patch-clamp.<br />La poursuite des travaux pour déterminer la nature moléculaire de la sélectivité des isoformes de SUR aux ouvreurs<br />pharmacologiques nous a permis de conclure que seul le faible encombrement de la Thr1253 de SUR2A, contre la Met<br />1290 de SUR1, serait le critère important pour l'activation pharmacologique des canaux KATP.<br />Nos travaux ont ensuite porté sur un domaine de la sous-unité SUR riche en acides aminés chargés négativement<br />(succession de 15 résidus glutamates ou aspartates) qui s'est avéré ne pas être impliquée dans la fonction du canal dans<br />notre système d'expression.<br />Nous avons étudié l'effet des ions Zn2+ et Cd2+ intracellulaires sur les canaux KATP et montré que ces ions peuvent activer<br />les canaux via leur liaison à SUR. Ce site de liaison reste encore à déterminer.<br />Nous avons enfin essayé de comprendre le rôle de chacun des domaines de liaison des nucléotides et nous avons pour cela<br />conçu des protéines SUR2A possédant des NBD identiques (NBD1-NBD1 et NBD2-NBD2) ou inversés (NBD2-NBD1).<br />Nos résultats suggèrent que (1) les NBD sont interchangeables (2) l'activation pas le Mg-ADP requiert les deux NBD (3)<br />l'action des ouvreurs est indépendante du NBD2.

L'ATP synthase ou ATPase de type F, ancrée aux membranes internes des mitochondries, est un complexe macromoléculaire qui utilise le gradient électrochimique généré par l'oxydation de petites molécules (NADH2, FADH2) dans les différents complexes de la chaîne respiratoire pour former l'ATP, vecteur énergétique universel. Le gradient électrochimique ou pmf est transformé en une énergie mécanique qui se traduit par le mouvement du rotor de l'ATP synthase dans un sens horaire vu depuis la membrane. La rotation de la sous-unité déforme successivement les trois sites catalytiques et permet ainsi la synthèse d'ATP. Dans certains cas, comme ceux de l'anoxie ou de l'hypoxie, le gradient électrochimique peut s'effondrer et l'ATP synthase hydrolyse alors l'ATP. Pour éviter cette hydrolyse futile, un petit peptide nommé IF1, régulateur spécifique des ATP synthases mitochondriales, vient s'insérer entre les sous-unités d'une interface catalytique et bloque instantanément le fonctionnement de l'ATPase. Cette inhibition est réversible puisque le peptide se décroche lorsque la membrane interne mitochondriale se réénergise. Dans ce travail de thèse, nous nous sommes intéressés à caractériser le mécanisme d'inhibition de l'ATPase de S.cerevisiae par son peptide endogène IF1 en s'appuyant essentiellement sur les quelques données structurales qui ont été publiées sur le peptide et sur le complexe inhibé IF1-F1-ATPase de B.taurus. Constitué de 63 acides aminés chez S.cerevisiae et 84 acides aminés chez B.taurus, IF1 est majoritairement structuré en hélice α. Les études menées par Elena Cabezón ont montré qu'IF1 possédait différentes formes dont la prédominance et l'activité dépendait essentiellement du pH. Chez B.taurus, il existe une forme inhibitrice dimérique prédominante à pH inférieurs à 6,5 et une forme tétramérique dont nous connaissons la structure 3D qui est non inhibitrice et prépondérante à pH supérieurs à 6,5. Chez S.cerevisiae, il existe une forme monomérique inhibitrice prépondérante à pH supérieur à 6,5 et une forme dimérique prédominante à pH inférieurs à 6,5 et dont le caractère inhibiteur ou non n'a pas encore été déterminé. Sur la base de la structure 3D de l'IF1 bovin, nous avons voulu identifier les régions de dimérisation du peptide de levure en utilisant la technique de marquage de spin couplée à de la spectroscopie RPE. En plaçant des marqueurs de spin (MTSL) en partie médiane (E33C) ou en C-terminale (L54C), nous avons pu favoriser l'interface de dimérisation plutôt en partie médiane du peptide. Ce travail est encore au stade embryonnaire et ne nous permet pas, à ce jour, d'identifier la zone exacte de dimérisation. Dans un deuxième volet, nous avons voulu caractériser le mécanisme d'inhibition d'un point de vue dynamique et nous avons pu en préciser les différentes étapes : reconnaissance, verrouillage et stabilisation. Pour cela, nous avons associé la mutagenèse sur le peptide et sur l'enzyme aux cinétiques d'inhibition. Nous avons tout d'abord évalué le rôle de plusieurs résidus situés en Cterminal de la sous-unité β, dans la région de l'interface α/ β qui se referme sur le peptide IF1, dans la reconnaissance moléculaire spécifique d'IF1 par l'ATPase mitochondriale. Nous avons ensuite montré que la partie N-terminale d'IF1 joue un rôle mineur dans la reconnaissance moléculaire mais son enroulement autour de la sous-unité constitue un loquet important dans la stabilisation du complexe inhibé. Enfin, la fermeture de l'interface catalytique sur IF1 crée une zone de contact entre la "bosse" de la sous-unité γ et la partie C-terminale de la sous-unité α qui constitue la dernière clef de blocage du peptide au sein de la F1-ATPase. Ce dernier point de fermeture est le seul qui n'implique aucun résidu du peptide IF1.