Дисертації з теми "Lipid signal"

Insulin resistance is the major metabolic defect in obesity and Type 2 diabetes. Increased lipid accumulation is strongly associated with insulin resistance. A significant component of insulin resistance is thought to be a reduced ability of insulin to activate the cascade of phosphorylation events that lead to the metabolic effects of this hormone. The broad aims of this thesis were to examine the effect of high-fat diets containing different fat subtypes on in vivo insulin signalling, under conditions normally used to detect whole body insulin resistance, and to compare the effects of acute and chronic lipid oversupply on insulin signalling in vivo. Time-course and dose-response effects of insulin stimulation on site-specific phosphorylation of key signalling proteins were studied in rat tissues in vivo, to establish an appropriate experimental system to examine the onset of activation of the insulin signalling pathway. It was determined that short insulin infusions with concurrent glucose infusion, similar to the beginning of a euglycaemic-hyperinsulinaemic clamp, significantly increased the phosphorylation of major intermediates of the insulin signalling pathway in important tissues of insulin action (skeletal muscle [RQ], liver [LIV] and white adipose tissue [EPI]). These experiments provided a platform to study insulin signalling under the same conditions used to study lipid-induced insulin resistance. The provision of diets enriched in polyunsaturated or saturated fatty acids (FA) resulted in the corresponding enrichment of these fat subtypes in rat plasma and tissues. However, the effects on insulin signalling were essentially the same. Both fat diets induced defects in sitespecific phosphorylation of insulin receptor substrate (IRS)-1 and protein kinase B (PKB) in RQ and LIV, but not EPI. This suggests that the amount of fat in the diet, rather than enrichment in a particular fat subtype, had a greater impact on the development of signalling defects and that the response to high-fat feeding was tissue-specific. A 3hr elevation of circulating FA (using a lipid/heparin infusion), to a level that is relevant in clinical Type 2 diabetes, impaired insulin-stimulated PKB phosphorylation with no significant effect on IRS-1 phosphorylation. This suggests that there may be differences in the way acute and chronic exposure to increased FA impair insulin signalling. The phosphorylation defects observed in both chronic and acute studies did not seem to be associated with activation of major stress signalling pathways (JNK and NFkB), which have been suggested to have a role in lipidinduced insulin resistance. In conclusion, these studies demonstrate that impaired IRS-1 and PKB phosphorylation do have a role in the reduced insulin action observed with lipid oversupply in vivo, because the changes were detected under similar conditions as those used to determine whole body insulin resistance.

L’anandamide (arachidonoiletanolammide, AEA), insieme con l’altro endocannabinoide 2arachidonoilglicerolo (2-AG), lega ed attiva i due recettori accoppiati a proteine G inibitorie, (GPCR), chiamati recettori dei cannabinoidi di tipo-1 (CB1R) e di tipo-2 (CB2R). I CB1R sono localizzati principalmente nel sistema nervoso centrale, ma sono anche espressi in tessuti periferici come le cellule immunitarie. Mentre i CB2R sono maggiormente espressi a livello periferico, ma sono anche presenti nel cervello. Quindi l’attivazione dei recettori CB1 o CB2 da parte dell’AEA e del 2-AG ha molti effetti sia a livello centrale che periferico. Queste azioni sono controllate attraverso un non ancora caratterizzato meccanismo cellulare, che regola il rilascio degli endocannabinoidi da precursori di membrana, il loro trasporto all’interno delle cellule, ed infine la loro eliminazione. L’agente chiave nella sintesi dell’AEA è la N-acilfosfatidiletanolammine (NAPE)-fosfolipasi D (NAPE-PLD), mentre la sua degradazione avviene attraverso un trasportatore di membrana per AEA (AMT), ed una fatty acid amide hydrolase (FAAH). Oltre i recettori CB, l’AEA lega anche il recettore dei vanilloidi di tipo 1 (TRPV1). Il 2-AG è invece rilasciato da lipidi di membrana per azione di una lipasi specifica, sn-1-diacilglicerolo (DAGL), ed è idrolizzato da una specifica monoacilglicerolo lipasi (MAGL). E’ stato dimostrato che il trasporto del 2-AG attraverso la membrana cellulare è saturabile ed energia-indipendente e che può occorrere attraverso lo stesso trasportatore dell’AEA. L’AEA ed il 2-AG, con altri congeneri, le proteine che legano, trasportano, sintetizzano ed idrolizzano questi lipidi formano il “sistema endocannabinoide”. I lipid rafts sono subdomini della membrana plasmatica che contengono alte concentrazioni di colesterolo e di sfingolipidi, e sono ben conosciuti modulatori dell’attivitità di un numero di GPCR. Infatti, essi modulano il segnale ed il “trafficking” in molti tipi cellulari. La crescente evidenza che i lipid rafts possono modulare il segnale degli endocannabinoidi ci ha spinto ad investigare il possibile effetto della loro integrità sui recettori CB, sul metabolismo dell’AEA e del 2-AG in cellule neuronali e del sistema immunitario. A tale scopo abbiamo utilizzato la metil-β-ciclo destrina (MCD), un depletore del colesterolo, un composto largamente utilizzato per distruggere l’integrità dei lipid rafts. Abbiamo utilizzato le cellule di glioma di ratto C6, perché esse hanno un ben caratterizzato sistema endocannabinoide. Abbiamo poi esteso lo studio alla linea cellulare di neuroblastoma umano CHP100, la quale ha la stessa abilità delle cellule C6 di metabolizzare l’AEA, ma sono prive del CB1R e quindi sono più sensibili all’attività pro-apoptotica dell’AEA. Abbiamo inoltre esteso lo studio agli enzimi che degradano e sintetizzano il 2-AG. Inoltre, abbiamo studiato la linea cellulare umana DAUDI, perché possiede l’AMT e l’enzima FAAH ed esprime un CB2R funzionale. Le cellule DAUDI, inoltre, regolano importanti funzione in cui sono coinvolti i lipid rafts, come la secrezione di esosomi e l’arresto della crescita indotta dai farmaci antitumorali. Inoltre abbiamo valutato l’effetto della deplezione e dell’arrichimento di colesterolo sul metabolismo degli endocannabinoidi. In conclusione, questo studio ha monitorato l’effetto dell’integrità dei lipid rafts sulle principali proteine che legano e metabolizzano l’AEA ed il 2-AG, sia in cellule neuronali che in cellule del sistema immunitario. Tale studio indica che il CB1R ed il trasportatore degli endocannabinoidi sono probabilmente localizzati all’interno dei lipid rafts a differenza del CB2R e delle altre proteine che compongono il sistema endocannabinoide.
Anandamide (arachidonoylethanolamide, AEA) and the other endocannabinoid 2-arachidonoylglycerol (2-AG) bind to and activate two G protein-coupled receptors (GPCR), namely type-1 (CB1R) and type-2 (CB2R) cannabinoid receptors. CB1R are localized mainly in the central nervous system, but are also expressed in peripheral tissues like immune cells. Conversely CB2R are predominantly expressed peripherally, but they are also present in the brain. Therefore, activation of CB1 or CB2 receptors by AEA or 2-AG has many central and peripheral effects. These actions are controlled through not yet fully characterized cellular mechanisms, that regulate the release of endocannabinoids from membrane precursors, their uptake by cells, and finally their intracellular disposal. The key agent in AEA synthesis is the N-acylphosphatidylethanolamines (NAPE)-hydrolyzing phospholipase D (NAPE-PLD), whereas degradation occurs through an AEA membrane transporter (AMT), and a fatty acid amide hydrolase (FAAH). Besides CB receptors, AEA binds also to type 1 vanilloid receptors (now called transient receptor potential channel vanilloid receptor subunit 1, TRPV1). On the other hand, 2-AG is released from membrane lipids by means of a sn-1-specific diacylglycerol lipase (DAGL), and is hydrolyzed by a specific monoacylglycerol lipase (MAGL). The transport of 2-AG through the cellular membrane has been shown to be saturable and energyindependent, and might occur through the same AMT that transports AEA. Altogether AEA and 2-AG, with other congeners, the proteins that bind, transport, synthesize and hydrolyze these lipids, form the “endocannabinoid system”. Lipid rafts are subdomains of the plasma membrane that contain high concentrations of cholesterol and glycosphingolipids, and are well-known modulators of the activity of a number of GPCR. In fact, they modulate signaling and membrane trafficking in many cell types. The growing evidence suggesting that lipid rafts might modulate the endocannabinoid signaling prompted us to investigate also the possible effect of lipid rafts integrity on CB receptors, on AEA metabolism in neuronal and immune cells and on the proteins that synthesize, transport and degrade 2-AG. We have used the methyl--cyclodextrin (MCD), a membrane cholesterol depletor that is widely used to disrupt the integrity of lipid rafts. We have chosen rat C6 glioma cells, because they have a well characterized endocannabinoid system. We extended the study to human CHP100 neuroblastoma cells, which have the same ability as C6 cells to metabolize AEA, but are devoid of CB1R and hence are more sensitive to the pro-apoptotic activity of AEA. We did not further extend this study to 2-AG and the enzymes that degrade and synthesize it, because 2-AG does not have pro-apoptotic activity toward C6 cells or CHP100 cells, in keeping with the observation that it does not activate TRPV1 receptors. Furthermore, we have chosen human DAUDI leukemia cells, because they have active AMT and FAAH, and express functional CB2R. On the other hand, in DAUDI cells lipid rafts regulate important functions like exosome secretion, or growth arrest induced by antitumor drugs. In addition, we checked for the first time the effect of membrane cholesterol depletion or enrichment on 2-AG metabolism in C6 cells and DAUDI cells. In conclusion, this study monitor the effect of lipid rafts integrity on all the major proteins that bind and metabolize AEA and 2-AG, both in neuronal and immune cells. The results point out that CB1R and endocannabinoid transporters are probably localized within lipid rafts, at variace with CB2R and the other proteins of the endocannabinoid system.

Thesis (Ph.D. in Toxicology) -- University of Colorado, 2005.
Typescript. Includes bibliographical references (leaves 141-156). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

The membrane proteins signal peptide peptidase, signal peptide peptidase like and presenilin are intramembrane aspartyl proteases located in the endoplasmic reticulum, plasma membrane and organelle. These membrane proteins are able to catalyze a hydrolytic reaction in a hydrophobic space. The downstream consequences of these reactions impact a variety of cellular functions such as cytokine production, inflammatory responses, embryogenesis, and immune system regulation. Additionally, the aspartyl proteases such as signal peptide peptidase and presenilin, a part of the γ-secretase complex, hydrolyze peptides leading to pathogen maturation and Alzheimer’s disease, respectively. Electron crystallography offers the unique aspect of studying membrane proteins in a near native state. Determining the structures of Haloarcula morismortui and Methanoculleus marisnigri JR1 signal peptide peptidases by electron crystallography may provide insight into how a hydrolysis reaction occurs in a hydrophobic environment and how the protein determines which transmembrane signal peptides to cleave. Additionally, structure determination may help answer questions regarding why human presenilin, part of the γ-secretase complex, incorrectly processes amyloid precursor protein into amyloid-beta peptides leading to Alzheimer’s disease. Such structural data may not only shed light on how amyloid precursor protein is processed but how other proteins are processed by signal peptide peptidase leading to immune responses, cell signaling, and pathogen maturation. In addition, structure-function data may have an impact on pharmaceutical drug designs that targets signal peptide peptidase, signal peptide peptidase like, and/or presenilin. To determine the structure of aspartyl proteases, two archaeal signal peptide peptidases were used for two-dimensional crystallization trials to be able to study their structure by electron crystallography. Haloarcula morismortui and Methanoculleus marisnigri JR1 signal peptide peptidases, both human signal peptide peptidase homologues, were recombinantly over-expressed and purified. During dialysis trials, various lipid-to-protein ratios, sodium chloride concentrations, temperatures, detergents and a variety of other variables were tested. Methanoculleus marisnigri JR1 signal peptide peptidase showed the most promising results in terms of crystallinity. Optimizing dialysis conditions, specifically narrowing the lipid to protein ratio, resulted in two-dimensional crystals. Ordered arrays measuring up to 200 nm x 200 nm were observed. These ordered arrays have been shown to be reproducible amongst multiple batches of purified Methanoculleus marisnigri JR1 signal peptide peptidase. Preliminary projection maps of negatively stained ordered arrays show unit cell dimensions of a = 178 Å, b = 160 Å, γ = 92.0 Å and a = 175 Å, b = 167 Å, γ = 92.0 Å. The monomer measurements are approximately 70 Å by 80 Å. This is the first time a signal peptide peptidase homologue has been crystallized by two-dimensional crystallization.

Les récepteurs couplés aux protéines G (GPCRs en anglais) représentent la famille de récepteurs intégrales de membrane plus vaste dans la majorité des cellules eucaryotes. Ils jouent un rôle clé dans la transduction de signal, ainsi que la compréhension de leur mécanisme de signalisation représente une des questions principales dans la biologie d'aujourd'hui. Dans la caractérisation du paysage énergétique de ces récepteurs à l'échelle atomique, les structures cristallographiques publiées pendant la décennie dernière par cristallographie aux rayons X représentent la percée scientifique majeure et donnent une contribution fondamentale dans la biologie structurelle de GPCRS. Ces structures représentent un point de départ précieux dans la compréhension du mécanisme de transduction de signal, en plaçant des structures dans l'ensemble conformationnel de ces récepteurs le long du processus d'activation. Pour compléter ce cadre de structures statiques qui correspondent aux états à basse l'énergie et fortement peuplés, une caractérisation de l'ensemble conformationnel et des barrières cinétiques qui sont associées est un point nécessaire et fondamentale. À ce but nous proposons une approche innovant avec la finalité d'observer le paysage conformationnel dynamique des GPCR et étudier la modulation de ces récepteurs par des ligands et des lipides, qui sont connus pour jouer un rôle clé dans la structure et les fonctions des protéines de membrane (e.g.). Un des outilles le plus approprié pour explorer les barrières cinétiques de GPCR c'est la résonance magnétique nucléaire (RMN) en solution. Pour tirer profit au mieux de cette technique, nous avons utilisé des sondes marqués 13CH3 immergées dans un environnement perdeuteré, qui constitue le marquage isotopique le plus approprié en RMN pour examiner les paysages conformationnels des protéines de grosses dimensionnes ou des complexes de protéines. Nous avons choisi Escherichia coli comme système d'expression pour sa capacité de pousser dans des conditions très hostiles comme des solutions 100%-D2O. Pour surmonter les difficultés habituellement rencontrées lors de l'expression des GPCRs, nous avons appliqué un protocole innovant qui cible l'expression de GPCRs directement aux corps d'inclusion. Ceci permet la production des bonnes quantités de protéines (jusqu’à 6 mg/litres de culture de pur 13CH3-u-2H-GPCRs). Une fois purifié, le récepteur est foldé en amphipols et transféré ensuite à une double couche lipidique appelée nanometric lipid bilayer ou nanodisc (NLB). De façon très important, les mesures pharmacologiques quantitatives indiquent que les récepteurs incorporés dans des NLBs après ce protocole sont stables et entièrement actifs dans les conditions des expériences de NMR.Les investigations par RMN conduites sur le GPCR en NLB ont donné lieu à une résolution jamais obtenue dans le domaine, grâce à la biochimie finement accordée et à la perdeuteration du récepteur. Selon les données obtenues, notre récepteur modèle, le récepteur 2 pour le leukotriene B4 (BLT2), est capable d'explorer plusieurs conformations différentes, même dans l'état pas lié aux ligands, y compris l'état actif. Ce paysage conformationnel est également modulé par des ligands et des lipides. Dans le cas spécifiques, nous avons observé que un incrément dans le contenu de stérol dans la membrane modifie la distribution des différents états conformationnels du récepteur, en favorisant l'état actif, qui indique une régulation allosteric positif du stérol sur l'activation de ce récepteur, comme confirmé aussi par les mesures de liaison du GTP à la protéine G. Cette propriété du stérol est probablement importante pour le contrôle de mécanisme de signalisation de GPCRs
G protein-coupled receptors (GPCRs) are the largest family of integral membrane protein receptors present in most eukaryotic cells. They play a key role in signal transduction and understanding their signalling mechanism represents one of the main issues in biology today. In the characterization of the energy landscape of these receptors, at the atomic scale, X-ray crystal atomic structures published during the last decade represent the major breakthrough and contribution in the structural biology of GPCRs. They represent a precious starting point in the understanding of the mechanism of signal transduction by placing structures in the conformational ensemble of these receptors along the activation pathway. To complete these static snapshots that correspond to low energy and highly populated states, a characterization of the whole conformational ensemble and associated kinetic barriers is fundamental to complete the picture. To this aim we proposed an innovative approach to observe GPCRs dynamic conformational landscape and how it is modulated by ligands and lipids, that are known to play a key role in membrane protein structures and functions (e.g.). One of the most appropriate tool to explore GPCR kinetic barriers is solution state NMR. To do so, we used 13CH3 probes immersed in a perdeuterated environment, the most appropriate isotope-labelling scheme to investigate conformational landscapes of large proteins or protein complexes with this spectroscopy. We chose Escherichia coli as expression system for its ability to grow in very hostile conditions like 100%-D2O solutions. In order to overcome the usual expression issues concerning GPCRs, we applied an innovative protocol which targets the expression directly to inclusion bodies. This allows the production of high amounts of proteins (up to 6 mg/litre of culture of pure 13CH3-u-2H-GPCRs). Once purified, receptors are folded in amphipols and then transferred to nanometric lipid bilayers or nanodiscs. Importantly quantitative pharmacological measurements indicate that receptors embedded in NLBs following this protocol are stable and fully active in the conditions of the NMR experiments. NMR investigation of a GPCR in a NLB gave rise to a resolution never achieved in the field thanks to a fine tuned biochemistry and a perdeuteration of the receptor. According to our data, the prototypical receptor, the leukotriene B4 receptor (BLT2), is able to explore multiple different conformations, even in the unliganded state, including the active state. This conformational landscape is further modulated by ligands and lipids. In particular, we observed that an increment in the sterol content of the membrane modifies the distribution of the different conformational states of the receptor in favour of the active one, indicating a positive allosteric regulation of the sterol on the activation of this receptor, as confirmed by GTP-to-G protein binding measurements. This property of the sterol is likely important for the control of the signalling properties of GPCRs

Morphogens of the Wnt and Hedgehog families are secreted signaling molecules that coordinate growth and patterning of many different tissues. Both, Wingless and Hedgehog spread across long distances in developing wing of Drosophila melanogaster. However, both proteins are covalently modified with lipid moieties. The mechanisms that allow long-range movement of such hydrophobic molecules are unclear. Like Wingles and Hedgehog, glycosylphosphatidylinositol (gpi)-linked proteins also transfer between cells with their lipid anchor intact. It has been speculated that gpi-linked proteins and lipid-linked morphogens travel together on a membranous particle, which was termed an argosome. As yet however, no functional link between argosome production and dispersal of lipid-linked proteins has been established. The topic of this thesis is to understand the cell biological nature of the argosome and thus contribute to understanding of morphogen gradient formation. To address the question of argosome biosynthesis, at least two models have been proposed. One possibility is that argosomes are membranous exovesicles with a complete membrane bilayer. Alternatively, argosomes might resemble lipoprotein particles that comprise on of a family of apolipoproteins scaffolded around a phospholipid monolayer that surrounds a core of esterified cholesterol and triglyceride. Lipid-modified proteins of the exoplasmic face of the membrane (like GFPgpi, Wingless or Hedgehog) might fit well into the outer phospholipid monolayer of such a particle. Here, I utilize biochemical fractionation to determine the sort of particle that lipid-linked proteins associate with. I show that Wingless, Hedgehog and gpi-linked proteins bind Drosophila lipoprotein particles in vitro, and colocalize with them in wing imaginal discs. Next, I use genetic means to address the functional importance of this association. I demonstrate that reducing Lipophorin levels in Drosophila larvae perturbs long-range but not shor-range Wingless and Hedgehog signaling, and increases the sequestration of Hedgehog by Patched. I propose that Lipophorin particles are vehicles for the long-range movement of lipid-linked morphogens and gpi-linked proteins.

Une étude statistique de l'organisation mondiale de la santé a révélé qu'un adulte sur six est concerné par des problèmes d'infertilité. Ce sujet majeur de nos sociétés est complexe, multifactoriel avec une évolution à l'échelle mondiale qui est difficile à évaluer. Il est donc essentiel de mener davantage de recherche pour mieux comprendre l'évolution de l'infertilité, mais aussi les mécanismes cellulaires et moléculaires conduisant à une bonne fertilité.Fortuitement, un crible génétique des enzymes responsables de la synthèse des acides gras dans des cellules spécialisées de la drosophile a déclenché un phénotype de stérilité. Ces cellules spécialisées que l'on nomme oenocytes sont essentielles aux métabolismes des acides gras et impliquées dans de nombreux processus tels que l'homéostasie lipidique, la protection contre la dessication et la communication phéromonale.Mes travaux montrent que la synthèse d'un ou plusieurs acides gras à très longue chaîne dans les oenocytes est indispensable à la fertilité des femelles et que le défaut de cette synthèse provoque une rétention des spermatozoïdes dans les organes de stockage, spermathèques et réceptacle séminal. J'ai montré que le phénotype de stérilité n'est pas lié à un défaut d'activité des spermatozoïdes et qu'ils fertilisent efficacement les ovocytes matures. En revanche mes résultats indiquent que les oeufs présentent un défaut d'activation empêchant leur développement.Chez les insectes l'activation de l'ovocyte mature qui permet le développement embryonnaire ne dépend pas de l'entrée du spermatozoïde comme chez les mammifères. Cette activation est déclenchée par un signal calcique lors du passage dans les voies génitales femelles. L'ensemble de mes résultats montrent pour la première fois qu'un signal lipidique extra-génital provoque l'activation des ovocytes matures permettant ainsi l'induction du développement embryonnaire
A statistical study by the World Health Organization revealed that one adult over six is affected by infertility problems. This major social issue is complex and multifactorial, with worldwide trends that are difficult to assess. It is therefore essential to carry out more research to better understand not only the evolution of infertility, but also the cellular and molecular mechanisms leading to efficient fertility.Serendipitously, we discovered that a genetic screen to enzymes responsible for fatty acid synthesis in specialized Drosophila cells provoked a sterile phenotype. These specialized cells, called as oenocytes, are essential for fatty acid metabolism, and are involved in numerous processes, including lipid homeostasis, protection against desiccation and pheromonal communication.My work shows that the synthesis of one or more very long-chain fatty acids in oenocytes is essential for female fertility, and that a defect in this synthesis causes spermatozoa to be retaintion in the storage organs, spermathecae and seminal receptacle. I have shown that the sterility phenotype is not linked to a defect in sperm activity, and that sperm fertilize mature oocytes efficiently. On the other hand, my results indicate that the eggs show an activation defect preventing their development.In insects, activation of the mature oocyte, which leads to embryonic development, is not dependent on sperm entry as in mammals. This activation is triggered by a calcium signal while the oocyte moves through the female genital tract. Taken together, my results show for the first time that an extra-genital lipid-signal triggers the activation of mature oocytes, thus enabling the induction of embryonic development

Das Außensegment der Stäbchenzelle ist aus einem Stapel von flachen Membransäckchen, den Diskmembranen, aufgebaut. Entlang dessen existiert ein Cholesterolgradient mit 24 mol% Cholesterol in den basalen Diskmembranen und 5 mol% in den apikalen. Das Außensegment enthält alle Proteine der Signaltransduktion. Der Photorezeptor Rhodopsin ist als integrales Membranprotein in die Diskmembran eingebettet. Das G-Protein Transducin und das Effektorprotein, die Phosphodiesterase (PDE), sind periphere Proteine mit Lipidankern und somit reversibel mit der Membranoberfläche assoziiert. Um den Einfluss des Cholesterolgehaltes der Diskmembranen auf diese drei Proteine zu untersuchen, wurden Diskmembranen mit unterschiedlichem Cholesterolgehalt präpariert (Simulation des Cholesterolgradienten). Die Untersuchungen zur transversalen Verteilung des Cholesterols in der Diskmembran ergaben eine schnelle Transmembranbewegung mit einer Halbwertzeit von weniger als einer Minute bei 35 °C. Desweiteren konnte gezeigt werden, dass es zu kopfgruppenspezifischen Wechselwirkungen von Cholesterol mit dem Phospholipid Phosphatidylcholin kommt. Cholesterol verschiebt das Meta I-Meta II-Gleichgewicht (nach Lichtaktivierung von Rhodopsin) auf die Seite von Meta I (inaktiv). In dieser Arbeit konnte jedoch gezeigt werden, dass durch die Anwesenheit des Transducins das Gleichgewicht vollständig auf die Seite von Meta II (aktiv) verschoben wird, da Transducin spezifisch die Meta-II-Form stabilisiert. Somit kann die verminderte Meta II-Bildung des Rezeptors in Diskmembranen mit hohem Cholesterolgehalt durch Transducin ausgeglichen werden. Lediglich die Geschwindigkeit der Transducinaktivierung ist verlangsamt. Durch den erhöhten Cholesterolgehalt werden die Membraneigenschaften für eine Bindung der beiden peripheren Proteine Transducin und PDE über deren Lipidanker optimiert. Somit kann die Signaltransduktion auch in den basalen Diskmembranen des Stäbchenaußensegmentes stattfinden.
The rod outer segment consists of a stack of flat membrane saccules called disc membranes. Along this stack a cholesterol gradient exists with 24 mol% cholesterol in the basal and only 5 mol% in the apical disc membranes. The outer segment contains all the proteins necessary for signal transduction. The photoreceptor rhodopsin as integral membrane protein is embedded in the disc membrane. The G protein transducin and the effector protein phosphodiesterase (PDE) are soluble proteins with lipid modifications, which are associated reversibly to the membrane surface. Disc membranes with different cholesterol contents were prepared to simulate the cholesterol gradient along the rod outer segment and to investigate the influence of disc membrane cholesterol content of these three proteins. Investigations of the transversal distribution of cholesterol in the disc membrane revealed a fast transmembrane movement with a half life of less than one minute at 35 °C. Further, head group specific interactions between cholesterol and phosphatidylcholine could be shown. The Meta I Meta II equilibrium after light activation of rhodopsin was shifted to the Meta I (inactive) site in membranes with high cholesterol. In this work it was shown that in the presence of transducin this equilibrium is shifted completely to the Meta II (active) site because transducin stabilizes specifically the Meta II form of the receptor. Hence the reduced Meta II formation in disc membranes with high cholesterol could be compensated by transducin. The speed of transducin activation is decelerated. By the increased cholesterol content membrane properties are optimized to the binding of transducin and PDE via their lipid modifications. Thus the signal transduction can take place also in disc membranes with high cholesterol.

A superexpressão de receptores HER-2 é anormalidade celular de grande relevância clínica no câncer de mama. Ela ocorre em aproximadamente 30% de carcinomas de mama incluindo lesões pré-neoplásicas e malignas, e está associada a prognóstico desfavorável. A hiperativação dos receptores HER-2, consequência natural de sua superexpressão, promove proliferação celular aberrante e tumorigênese. Admite-se que a ativação e envio de sinais via HER-2 possa acontecer quando estes receptores se encontram em compartimentos específicos da membrana celular, os lipid rafts. Assim, um número maior de HER-2 poderia implicar em maior quantidade de lipis rafts. Para testar essa hipótese, usamos modelo de transformação oncogênica que nos permitiu avaliar, especificamente, os efeitos da superexpressão de HER-2 e identificar a quantidade de lipid rafts. Para isso utilizamos a linhagem celular HB4a, derivada de célula epitelial luminal do tecido mamário humano normal com baixa expressão de HER-2; e a linhagem HB4aC5.2, um clone derivado da HB4a, que superexpressa receptores HER-2. Nas células HB4aC5.2, a superexpressão de HER-2 foi acompanhada pelo aumento dos lipid rafts na membrana celular, bem como, hiperativação de sinais de sobrevivência, proliferação (aumento da ativação de proteínas Akt e Erk1/2, respectivamente), e taxa de proliferação celular duas vezes mais rápida que a linhagem normal HB4a. Adicionalmente, a superexpressão de HER-2 foi associada com aumento da lipogênese celular (fenótipo lipogênico), dependente do aumento de ativação da enzima FASN e da superexpressão de DEPTOR. A FASN é responsável pela síntese de palmitato, utilizado para formação de lipid rafts. A superexpressão de DEPTOR, por modular a atividade transcricional de PPAR?, pode evitar a lipotoxicidade do excesso de palmitato. Além disso, DEPTOR, por sua capacidade em reduzir atividade do complexo mTORC1, contribui para sobrevivência celular dependente da proteína Akt. Em continuidade, consideramos, como segunda hipótese, que a desestruturação de lipid rafts poderia influenciar negativamente a ativação dos receptores HER-2. Para isso tratamos, as mesmas linhagens celulares anteriormente descritas, com ácido docosaexaenoico (DHA), um tipo de ácido graxo ômega-3. Nossos resultados mostraram que, nas células HB4aC5.2, o tratamento com DHA desestruturou os lipid rafts, inibiu a sinalização iniciada pelos receptores HER-2 ( diminuição da ativação das proteínas Akt, Erk1/2, FASN, atividade transcricional de PPAR? e expressão de DEPTOR) e reverteu o fenótipo lipogênico. Adiciona-se que essas modificações celulares e moleculares foram acompanhadas por indução significativa de morte e apoptose. As mesmas alterações não foram observadas nas células normais HB4a. Em conclusão, o presente estudo reforça a associação entre a presença de HER-2 e lipid rafts. Adicionalmente aponta que a desestruturação de lipid rafts por DHA reduz a sinalização de HER-2. Por fim, sugere que distúrbios em lipid rafts, induzidos por DHA, possam representar ferramenta útil no controle da sinalização aberrante deflagrada pelos receptores HER-2, e aponta potencial terapêutico na suplementação de DHA para quimioprevenção e tratamento do câncer de mama HER-2 positivo.
HER-2 receptor overexpression is a cellular abnormality of great clinical significance in breast cancer. It is described in approximately 30% of breast carcinomas, including preneoplasic and malignant lesions, and is associated with poor prognosis. Hyperactivation of HER-2 receptors, a natural consequence of its overexpression, promotes aberrant cell proliferation and tumorigenesis. For signal activation and transduction to occur, HER-2 must be localized in specific compartments in the cell membrane: the lipid rafts. Therefore, we hypothesize that a greater number of HER-2 receptors could indicate a greater quantity of lipid rafts. To test this, we used an oncogenic transformation model that specifically allowed assessment of the effects of HER-2 overexpression and identification of the quantity of lipid rafts: an HB4a cell line derived from normal human breast tissue luminal epithelial cells with low HER-2 expression, and an HB4aC5.2 cell line, a clone derived from HB4a that overexpresses HER-2 receptors. In the HB4aC5.2 cells, HER-2 overexpression was accompanied by an increase in lipid rafts in cell membranes as well as hyperactivation of survival signals, proliferation (increased activation of the proteins Akt and ERK1/2, respectively), and an increased rate of proliferation, compared to the normal HB4a line. In addition, HER-2 overexpression was associated with increased cellular lipogenesis (lipogenic phenotype), dependent on the increased activation of the FASN enzyme and the overexpression of DEPTOR. FASN is responsible for the synthesis of palmitate, used to synthesize lipid rafts. Overexpression of DEPTOR by modulating PPAR? transcriptional activity, may avoid lipotoxicity from excess palmitate. Moreover, DEPTOR, with its ability to reduce mTORC1 complex activity, contributes to cell survival dependent on Akt. To continue, we considered as a second hypothesis that the disruption of lipid rafts could negatively influence HER-2 receptor activation. For this, we treated the same cell lines described above with docosahexaenoic acid (DHA), a omega-3 fatty acid. Our results showed that in HB4aC5.2 cells DHA treatment disrupted the lipid rafts, inhibited signaling initiated by HER-2 receptors (reduced activation of Akt, ERK1/2, and FASN proteins, PPAR? transcriptional activity, and DEPTOR expression) and reversed the lipogenic phenotype. In addition, these cellular and molecular changes were accompanied by a significant induction of apoptosis and death. The same changes were not observed in normal HB4a cells. In conclusion, the present study reinforces the association between HER-2 presence and lipid rafts. It also indicates that the disruption of lipid rafts by DHA reduces HER-2 signaling. Finally, it suggests that DHA-induced disturbances in lipid rafts may represent a useful tool in controlling aberrant signaling triggered by HER-2 receptors, and indicate therapeutic potential in DHA supplementation for chemoprevention and treatment of HER-2 positive breast cancer.

Le TNF alpha (Tumor Necrosis Factor alpha) est une cytokine pro-inflammatoire existant sous deux formes moléculaires, une forme transmembranaire étant clivée au niveau de la membrane pour générer une forme soluble. Le TNF alpha est impliqué dans de nombreux processus physiologiques et physiopathologiques dont l'athérosclérose. Au sein de la plaque, le TNF alpha soluble semble jouer un rôle majeur : des souris génétiquement modifiées et exprimant exclusivement une forme membranaire non clivable du TNF alpha développent des plaques d'athérosclérose dont l'état inflammatoire est significativement réduit par rapport à celles des souris sauvages (Canault et al. , 2004). Le clivage de la forme membranaire du TNFalpha est médié par la métalloprotéase ADAM-17 (A Disintegrine and Metalloproteinase-17). A ce jour, la régulation de l'activité de clivage d'ADAM-17 est peu documentée. Nous avons émis l'hypothèse que la localisation membranaire d'ADAM-17 et de ses substrats participait à cette régulation. Nous montrons que la forme mature d'ADAM-17 est contenue dans des domaines particuliers de la membrane, les radeaux lipidiques (Tellier et al. , 2006), et que cette localisation conditionne son activité. Nous avons également montré que des modifications de l'homéostasie du cholestérol des cellules par des HDLs conduisaient à une perturbation du clivage des substrats d'ADAM-17 (Tellier et al. , 2007). Le TNF alpha induit de multiples voies de signalisation dans les cellules de la plaque d'athérosclérose, conduisant, entre autres, à la prolifération des CML (cellules musculaires lisses). La littérature montre que le TNF alpha induit l'activation de sphingomyélinases (SMase) neutres et que le rôle mitogène des SMases implique les métalloprotéinases MMP-2 et MT1-MMP (Auge et al. , 2004 ; Nègre-Salvayre et al. , 2005). Nous avons posé l'hypothèse que le TNF alpha pouvait induire la prolifération cellulaire par une voie impliquant l'activation des métalloprotéases et des sphingomyélinases neutres. Nos résultats montrent que le TNFalpha stimule la prolifération des cellules musculaires lisses et des fibroblastes par activation séquentielle de la pro-protéine convertase furine, des métalloprotéases MT1-MMP et MMP-2, puis de la voie sphingomyéline/céramide (Tellier et al. , 2007). En conclusion, nos résultats apportent des données nouvelles sur la régulation de la biodisponibilité du TNF alpha ainsi que sur son effet pro-athérogène via l'activation d'une voie mitogène de stress
TNF alpha (Tumor Necrosis Factor alpha) is a pro-inflammatory cytokine existing in two molecular forms, a transmembrane form cleaved to produced a soluble form. TNF alpha is implicated in numerous physiological and physiopathologicals processes such as atherosclerosis. Within the lesions, soluble TNF alpha seems to play a major role : genetically modified mice expressing exclusively a transmembrane form of TNF alpha develop atherosclerosis lesions with an inflammatory state which is significantly reduced compared to lesions of wild type mice (Canault et al. , 2004). Cleavage of TNF alpha transmembrane form is due to the metalloproteinase ADAM-17 (A Disintegrine and Metalloproteinase-17). Today, regulation of ADAM-17 cleavage activity is poorly documented. We have voiced hypothesis that the membrane localization of ADAM-17 and its substrates was implicated to its regulation. We show that ADAM-17 mature form is localized into membrane particular domains, the lipid rafts (Tellier et al. , 2006) and that this localization determined its activity. We also show that cell cholesterol homeostasis modifications by HDLs lead to a ADAM-17 substrates cleavage perturbations (Tellier et al. , 2007). TNF alpha induce number signaling pathways in atherosclerosis plaques cells, leading to several effects including SMC (smooth muscle cells) proliferation. Literature show that TNF alpha active neutral sphingomyelinase (nSMase) and that nSMase mitogene effect involve MMP2 and MT1-MMP (Auge et al. , 2004 ; Nègre-Salvayre et al. , 2005). We have postulated that TNF alpha could induce cell proliferation by a pathway implicating metalloproteinases and neutral SMase activation. Our results show that TNF alpha induced mesenchymal cells proliferation is due to pro-protein convertase furin, MT1-MMP ad MMP2 metalloproteinases sequential activation followed by sphingomyeline/ceramide pathway (Tellier et al. , 2008). In conclusion, our results give new data on the TNF alpha biodisponibility regulation as on pro-atherogene effect via stress mitogene pathway activation

Thesis: Ph. D., Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Department of Earth, Atmospheric, and Planetary Sciences; and the Woods Hole Oceanographic Institution), 2016.
Cataloged from PDF version of thesis.
Includes bibliographical references.
The role of oxylipins in ocean biogeochemistry was investigated using microcosm amendment experiments, environmental lipidomics, and culture based studies. Oxylipins are a bioactive class of secondary metabolites produced by diatoms and other eukaryotic phytoplankton. Previous research has focused mainly on one class of oxylipins, polyunsaturated aldehydes (PUAs), and their impacts on copepods. And few studies have looked at the impacts of oxylipins in situ. Here I show that oxylipins have the potential to impact carbon flux attenuation, oxylipin production in situ is linked to diatom bloom decline and viruses, and oxylipins deter microzooplankton grazing. Sinking particles collected in the North Atlantic were determined to be hot spots for PUAs with concentrations in the micromolar range. Natural particle associated microbial communities exhibited a dose dependent response to PUAs. Stimulatory PUA concentrations ranged from 1-10 ptM, resulting in enhanced remineralization of organic matter by particle associated microbes. Thus, PUAs produced during bloom decline may lead to greater flux attenuation and nutrient recycling. A novel lipidomics approach was applied along a cruise track in the California Coastal System revealing that canonical diatom free fatty acids and oxylipins dominated the dissolved lipidome and oxylipin abundance was correlated with diatom bloom demise as assessed by phaeophytin and biogenic Si. RNA viruses were likely the cause of diatom bloom demise and may have induced oxylipin production. The link between viruses and oxylipins represents a new infochemical signaling pathway in the ocean. Many oxylipins that are novel to the marine environment were also identified. The dissolved lipidome was sampled during grazing experiments with the microzooplankton grazer Oxyrrhis marina and both wild type Phaeodactylum tricornutum and a chronically stressed, transgenic strain (PtNOA). Grazing was suppressed in the PtNOA treatments compared to the WT, likely due to upregulation of small unknown lipophilic molecules. This suggests that cellular stress and oxylipin production may deter microzooplankton grazing in the environment potentially altering the transfer of energy through the microbial food web. By employing interdisciplinary approaches, we have learned that oxylipins production in situ is linked to bloom decline and the bioactivity of these compounds has significant implications for ocean biogeochemical cycles.
by Bethanie Rachele Edwards.
Ph. D.

It was suggested that under heat stress the accumulation of denatured proteins alone triggers the expression of heat shock proteins. However, earlier research suggested that during abrupt temperature fluctuations membranes represent the most thermally-sensitive macromolecular structures. The aim of this thesis to confirm experimentally for the membrane sensor theory in mammalian cells and to explore the mechanisms behind membrane lipid structural reorganizations. The main results are as follows: (i) I provide the first evidence that heat-analogous, chemically-induced membrane perturbation of K562 erythroleukemic cells is indeed capable of activating heat shock protein formation at the growth temperature, without causing measurable protein denaturation; (ii) I showed that the membrane fluidizer benzyl alcohol acts as a chaperone-inducer also in B16(F10) melanoma cells. Furthermore, following both alcohol and heat treatments, condensation of ordered plasma membrane domains was detected by fluorescence microscopy; (iii) lipidomic fingerprints revealed that stress achieved either by heat or benzyl alcohol resulted in pronounced and highly specific alterations of membrane lipids in B16(F10) cells. The loss in polyenes with the concomitant increase in saturated lipid species was shown to be a consequence of activation of phospholipases. The accumulation of lipid species with raft-forming properties may explain the condensation of ordered plasma membrane domains detected previously; (iv) with Laurdan two-photon microscopy it was demonstrated that, in contrast to the formation of ordered domains in surface membranes, the molecular disorder is significantly elevated within the internal membranes of cells preexposed to mild heat stress. These results were compared with those obtained by other probes and visualisation methods. It was found that the structurally different probes revealed substantially distinct alterations in membrane heterogeneity. The results highlight that even subtle changes in membrane microstructure may play a role in temperature sensing and thermal cell killing and, therefore, could have potential in treatment of several diseases.

Ce travail etaient est centre sur l'etude et la quantification du signal rmn dans certaines conditions in vivo. Une approche numerique de type monte carlo a ete utilisee. Une premiere etude porte sur la mesure in vivo du coefficient apparent de diffusion (cad) de lipides mobiles detectes dans des tumeurs cerebrales et confines dans des micro-domaines. Le modele numerique permet d'estimer la taille des micro-domaines sur la base du cad mesure. Un diametre de 10 a 12 m a ete trouve, valeur qui concorde avec les etudes par microscopie. Une seconde etudes porte sur la quantification des effets de la desoxygenation du sang sur la baisse du cad observee experimentalement lors de l'etude de l'ischemie. Une double etude numerique et experimentale nous a permis de conclure que la desoxygenation du sang n'est pas la principale cause de la baisse du cad. L'approche numerique est basee sur un modelisation du tissu cerebral prenant en compte la diffusion des molecules d'eau, le reseau vasculaire et les gradients internes generes autour ce dernier. Une troisieme etude porte sur la quantification du contraste bold (blood oxygenation level dependent) utilise en imagerie fonctionnelle. Differents parametres lies au secteur vasculaire, a la diffusion des molecules d'eau, et a la sequence d'impulsions ont ete considere. A partir de simulations basees sur une modelisation du tissu cerebral, des equations analytiques de la vitesse de relaxation de l'aimantation transversale en fonction des differents parametres d'interets ont ete. Dans ce travail, nous avons developpe un outil numerique aidant a la quantification du signal rmn et facilement adaptable aux diverses problematiques rencontrees dans differents secteurs de la rmn (diffusion, imagerie fonctionnelle).

Over the last decade, the innate immune system has been the subject of extensive research. Often overlooked by the robustness and specificity of the adaptive immune system, the innate immune system is proving to be just as complex. The identification of several families of pattern recognition receptors (PRRs) has revealed an ancient yet multifaceted system of proteins that are responsible for initiating host defense. A wide array of pathogens, from virus to bacteria, is detected using this assortment of receptors. One such family, the Toll-like receptors (TLRs), has been at the forefront of this research. To date, 10 TLRs have been described in the human genome. Activation of TLRs leads to the induction of immune-related genes that ultimately control the response of the host. However, the signaling pathways emanating from activated TLRs and other PRRs are not fully understood. In particular, the pathway leading to the activation of interferon regulatory factor 3 (IRF3), a transcription factor crucial for the induction of type I interferon, remains undefined. IRF3 activation occurs as the consequence of viral infection and through the activation of TLRs 3 and 4 by dsRNA and lipopolysaccharide (LPS), respectively. The focus of this research is to describe components of the IRF3 activation pathway, partly through the analysis of TLR signal transduction. IRF3 normally resides in the cytoplasm of cells. Upon infection with certain viruses and bacteria, IRF3 is activated though phosphorylation at its C-terminus. Phosphorylated IRF3 homodimerizes and associates with co-activators CBP-p300. After translocating to the nucleus, the activate IRF3 complex induces the activation of type 1 interferon and interferon related genes. Little is known about the pathways that lead to the activation of IRF3, especially the kinases involved. In this study we report that the non-canonical IкB kinase homologues, IкB kinase epsilon (IKKε) and TANK-binding kinase-1 (TBK1), which were previously implicated in NF-кB activation, are also essential components of the IRF3 signaling pathway. In particular, mouse embryonic fibroblasts from TBK1 deficient mice fail to activate IRF3 in response to both viral infection and stimulation with LPS or poly (IC), a dsRNA analog. Thus, both IKKε and TBK1 play a critical role in innate immunity and host defense. In addition to viral infection, IRF3 activation also occurs via the activation of TLR3 and 4. TLRs signal through a subfamily of Toll-IL-1-Resistance (TIR) domain containing adapter molecules. One such adapter, MyD88, is crucial for all TLRs, with the exception of TLR3. MyD88 participates in a signal transduction pathway culminating in the activation of the transcription factor NF-кB. Studies from MyD88-deficient mice reveal that both TLR3 and 4 still are capable of activating NF-кB, although with slightly delayed kinetics. Another aspect of the MyD88-independent signal transduction pathway is the activation of IRF3. A second TIR domain containing adapter molecule called Mal/Tirap was discovered and originally thought to mediate the MyD88-independent pathway. However, Mal-deficient mice were found to be defective in both TLR2 and 4 mediated NF-кB activation. We hypothesized that other TIR domain containing adapters could mediate this MyD88-independent pathway of TLR3 and 4 leading to the activation of IRF3. Two additional TIR adapters were discovered, TRIF and TRAM. TRIF was shown to mediate TLR3 signal transduction. In this study, we report that both TRIF and TRAM mediate the activation of the MyD88-independent pathway in response to LPS/TLR4 activation. Unlike any of the other known TIR domain containing adapters, TRAM appears to be restricted to the LPS/TLR4 activation pathway while TRIF plays a role in both TLR3 and TLR4 pathways leading to IRF3 target gene expression. Our studies revealed that TRAM could be acting upstream of TRIF in the LPS/TLR4 pathway. To this end, we sought to determine the localization of TRAM within the cell. We found that TRAM localizes to the plasma membrane. TRAM localization is the result of myristoylation since mutation of the predicted myristoylation site (G2A) resulted in the re-distribution of TRAM from the membrane into the cytoplasm. Reconstitution of TRAM-deficient macrophages with TRAM G2A is unable to rescue LPS/TLR4 signal transduction. Thus, myristoylation and membrane association of TRAM are critical for LPS/TLR4 signal transduction. The data generated in this dissertation extends our understanding of the signaling pathways of the innate immune system. Indeed, the molecules and pathways described herein could prove to be beneficial targets for ameliorating symptoms of disease, both autoimmune and pathogen-associated. Finally, the research described here will spur further insight into the complex signaling pathways of a once ignored arm of the immune system.

Membrane rafts are highly dynamic heterogeneous sterol- and sphingolipid-rich micro-domains on cell surfaces. They are generally believed to provide residency for cell surface molecules (e.g., adhesion and signaling molecules) and scaffolding to facilitate the functions of these molecules such as membrane trafficking, receptor transport, cell signaling, and endocytosis. Using laser scanning confocal microscopy and reflection interference microscopy (RIM), we studied the spatial and temporal distributions of membrane rafts and surface receptors, signaling molecules, and cell organelles during the formation of phagocytic contact areas. K562 cells, which naturally express CD32A, a cell surface receptor for the Fc portion of Immuno-globulin g (IgG), was chosen as a model for neutrophils. An opsonized target was modeled using a glass supported lipid bilayer reconstituted with IgG. CD32A was found to cluster and co-localize with membrane rafts. Placing the K562 cells on the lipid bilayer triggered a process of contact area formation that includes binding between receptors and ligands, their recruitment to the contact area, a concurrent membrane raft movement to and concentration in the contact area, and transport of CD32A, IgG, and membrane rafts to the Golgi complex. Characterization of these processes was performed using agents known to disrupt detergent resistant membranes (DRMs), dissolve actin microfilaments, and inhibit myosin motor activity, which abolished the CD32A clusters and prevented the contact area formation. The relevance to phagocytosis of contact area formation between K562 cells and lipid bilayers was demonstrated using micro-beads coated with a lipid bilayer reconstituted with IgG as the opsonized target instead of the glass supported planar lipid bilayer. Disruption of membrane rafts, salvation of the actin cytoskeleton, and inhibition of myosin II activity were found to inhibit phagocytosis. Here we have provided evidence that membrane rafts serve as platforms that are used to pre-cluster CD32A and transport CD32A along the actin cytoskeleton to the site of phagocytic synapse formation, followed by internalization to the Golgi complex.

The FYVE domain is a lipid binding domain found in approximately 27 different mammalian proteins. It specifically interacts with the lipid, PI(3)P, which is enriched on early endosomes. Consequently, many FYVE domain-containing proteins localize to the endosome, however the ability of FYVE domains to target to endosomal membranes is variable, despite high sequence conservation. Here we describe the structural requirements necessary for endosomal localization and liposome avidity. As FYVE domains are lipid binding domain, many FYVE domain-containing proteins have been implicated in membrane trafficking. We performed an RNAi screen of FYVE domain-containing proteins to identify general regulators of endocytosis in Caenorhabditis elegans. In this screen, we identified the EEA1, a known regulator of endocytosis and two novel genes: WDF2 and KIAA1643. Initial characterization of WDF2 suggest that its function is conserved in humans. Of all the FYVE domain-containing proteins, we have been particularly interested in SARA (Smad Anchor for Receptor Activation); a protein implicated in the TGFβ signaling pathway. This protein contains a binding domain for the TGFβ mediated transcription factor, Smad2/3, and a FYVE domain. It was the presence of the FYVE domain, an endosomal targeting signal, in SARA that lead us to hypothesize that endocytosis might be a necessary step in TGFβ signaling. SARA localizes to the early endosome; the TGFβ receptors also internalize into these endosome. When this internalization is prohibited, there is correlative decrease in Smad2/3 phosphorylation, Smad2 nuclear translocation and TGFβ mediated transcription. Overexpression of a dominant negative SARA construct and SARA siRNA oligonucleotides inhibit TGFβ signaling. We conclude that TGFβ receptor signaling to Smad2/3 occurs on the endosome and this signaling requires SARA. Receptor mediated endocytosis has been classically thought of as an important mechanism for attenuating signaling pathways. We have redefined the role of endocytosis to include the necessary and positive regulation of specific signaling pathways. We have also extended our insights into the biological role of the endosome as a compartment specialized for the assembly and propagation of specific extracellular signals.

The FYVE domain is a lipid binding domain found in approximately 27 different mammalian proteins. It specifically interacts with the lipid, PI(3)P, which is enriched on early endosomes. Consequently, many FYVE domain-containing proteins localize to the endosome, however the ability of FYVE domains to target to endosomal membranes is variable, despite high sequence conservation. Here we describe the structural requirements necessary for endosomal localization and liposome avidity. As FYVE domains are lipid binding domain, many FYVE domain-containing proteins have been implicated in membrane trafficking. We performed an RNAi screen of FYVE domain-containing proteins to identify general regulators of endocytosis in Caenorhabditis elegans. In this screen, we identified the EEA1, a known regulator of endocytosis and two novel genes: WDF2 and KIAA1643. Initial characterization of WDF2 suggest that its function is conserved in humans. Of all the FYVE domain-containing proteins, we have been particularly interested in SARA (Smad Anchor for Receptor Activation); a protein implicated in the TGFβ signaling pathway. This protein contains a binding domain for the TGFβ mediated transcription factor, Smad2/3, and a FYVE domain. It was the presence of the FYVE domain, an endosomal targeting signal, in SARA that lead us to hypothesize that endocytosis might be a necessary step in TGFβ signaling. SARA localizes to the early endosome; the TGFβ receptors also internalize into these endosome. When this internalization is prohibited, there is correlative decrease in Smad2/3 phosphorylation, Smad2 nuclear translocation and TGFβ mediated transcription. Overexpression of a dominant negative SARA construct and SARA siRNA oligonucleotides inhibit TGFβ signaling. We conclude that TGFβ receptor signaling to Smad2/3 occurs on the endosome and this signaling requires SARA. Receptor mediated endocytosis has been classically thought of as an important mechanism for attenuating signaling pathways. We have redefined the role of endocytosis to include the necessary and positive regulation of specific signaling pathways. We have also extended our insights into the biological role of the endosome as a compartment specialized for the assembly and propagation of specific extracellular signals.

Pain signaling involves transmission of nociceptive stimuli in the spinal cord where a critical balance between excitatory and inhibitory inputs determines the response to noxious stimuli. The neuropeptide, substance P (SP), mediates transmission of pain in part by binding to the tachykinin receptor (NK-1R) in the dorsal horn (DH) of the spinal cord. One of SP’s downstream effects is to modulate N-type Ca2+(N-) channels. While phospholipid breakdown is a part of the inflammatory process that accompanies tissue damage, the role of this metabolic pathway has not been completely described with respect to N-channel modulation during pain signaling. Despite the incomplete understanding of this modulation, pharmacological antagonists of both NK-1R and N-channels have been used to treat pain. In Chapter II, using whole-cell patch clamp recording techniques, the SP signaling cascade that mediates inhibition of recombinant N-channel activity was characterized. By adopting a pharmacological approach, I show that this pathway resembles the slow pathway that was earlier described for modulation of N-current by the M1 muscarinic receptor (M1R). M1R couples to Gq to stimulate phospholipid breakdown. Together with previous observations, the data presented in this chapter provide evidence for involvement of the extracellular receptor kinase (ERK1/2), phospholipase A2 and release of phospholipid metabolites in the modulation of N-current by SP. Overall, this chapter shows that phospholipid metabolism involved in modulation of N-currents is not specific to M1Rs but that other Gq-coupled receptors may also modulate N-currents via the same signal transduction pathway. In Chapter III, enhancement of N-current by SP was studied as part of a collaborative project to understand current enhancement that occurs when a palmitoylated accessory CaVβ2a subunit is co-expressed with the pore-forming subunit CaV2.2 and the accessory subunit α2δ-1. When CaVβ3 is present, SP inhibits N-current as described in Chapter II. However, when palmitoylated CaVβ2a is co-expressed with CaV2.2 (and α2δ-1), current enhancement is observed at negative test potentials, demonstrating that both M1Rs and NK-1Rs exhibit the same profile of N-current modulation. This change in modulation by muscarinic agonists is not observed in the presence of a depalmitoylated CaVβ2a. However a chimeric CaVβ2aβ1b subunit that contains the palmitoylated N-terminus from CaVβ2a confers enhancement. Normally expression of the β1b subunit resulted in current inhibition. These findings indicated that the palmitoylated CaVβ2a participates in enhancement of current. Our data support a model where inhibition dominates over enhancement; when inhibition is blocked, enhancement may be observed. Lastly, we show that N-current inhibition by SP is minimized when exogenous palmitic acid is applied to cells co-expressing CaVβ3 subunits with N-channels. These results indicate that the presence of palmitic acid can prevent N-current inhibition when SP is applied most likely by interacting with CaV2.2. We propose a model where palmitic acid occupies the inhibitory site and serves to antagonize inhibition by a lipid metabolite, which is most likely arachidonic acid. The CaVβ2a protein seems to have a role in positioning the palmitoyl groups near CaV2.2. This chapter provides a new role for protein palmitoylation where the palmitoyl groups of CaVβ2a are both necessary and sufficient to block inhibition of another protein: CaV2.2. In Chapter IV, I probe the role of the relative orientation of CaVβ2a and the pore-forming subunit of the N-channel in N-current modulation. Evidence is presented that shows that not just the presence of a palmitoylated CaVβ2a is necessary, but the relative orientation of CaVβ2a to CaV2.2 is critical for blocking inhibition. Using N-channel mutants that cause a change in the orientation of CaVβ2a relative to CaV2.2, I show that the block of inhibition is disrupted; inhibition by the slow pathway is rescued. These findings further support my model that the palmitoyl groups of CaVβ2a normally reside in a specific location that overlaps with the slow pathway inhibitory site on CaV2.2. Lastly I present data showing that the enhancement of N-current, observed when palmitoylated CaVβ2a is present, occurs via the slow pathway. In Chapter V the effect of CaVβ’s orientation on N-channel modulation by the dopamine D2 receptor is tested. In this form of modulation, inhibition is rapid and voltage-dependent. The signaling pathway is membrane-delimited since Gβγ, released after receptor stimulation, directly interacts with the N-channel at a site that overlaps with a high affinity binding site for CaVβs. While N-currents are modulated by this pathway, the deletion mutants show aberrant membrane-delimited modulation. The findings in this chapter further underscore the importance of proper positioning of CaVβ to CaV2.2 for eliciting proper N-current modulation after GPCR stimulation. Overall, the data presented in this dissertation provides a mechanistic approach into examining modulation of N-current by different GPCRs via two different signaling pathways as well as the role CaVβ subunits serve in each modulatory pathway.

Nouvelles voies de modulation des canaux calciques de type T.Grâce à leur rôle dans l'excitabilité cellulaire et l'homéostasie calcique, les canaux calciques de type T participent à différentes fonctions physiologiques telles que le sommeil ou le contrôle du rythme cardiaque et de la pression artérielle. Ils sont également impliqués dans certaines pathologies comme la douleur ou l'épilepsie. La régulation de l'activité des canaux de type T est encore mal connue et c'est l'enjeu de cette thèse. Dans une première partie, nous avons caractérisé l'effet de certains lipides endogènes sur ces canaux, en particulier les métabolites de l'acide arachidonique, et identifié le 5,6-EET comme un nouveau bloqueur des canaux de type T. Nous avons ensuite évalué l'existence d'un site de liaison des lipoamino acides sur les canaux de type T à l'aide d' d'expériences de compétitions réalisées avec un inhibiteur spécifique de ces canaux, la molécule TTA-A1.Dans une deuxième partie, nous avons étudié la régulation par le calcium des canaux de type T. Nous avons montré que l'entrée de calcium par les canaux T eux-mêmes ou par activation des récepteurs P2X4 et NMDA induisait une inhibition du courant de type T. Le calcium activerait une phosphatase qui provoquerait un déplacement de la courbe d'inactivation à l'état stable vers les potentiels négatifs, réduisant ainsi la disponibilité des canaux. Ce phénomène d'inhibition du courant lié à l'entrée du calcium pourrait être un mécanisme de rétrocontrôle négatif limitant l'entrée de calcium dans la cellule afin d'éviter une toxicité provoquée par une concentration de calcium intracellulaire trop élevée. Suite à cette inhibition, lorsque l'entrée de calcium est arrêtée, le courant augmente. Cette augmentation semble être due à l'intervention de la protéine kinase Pak qui rendrait les canaux de type T disponibles.En conclusion, nous avons identifié et caractérisé deux nouveaux mécanismes endogènes de régulation des canaux de type T : une modulation par les lipides, et une modulation par les calcium et la protéine kinase Pak
New pathways of regulation of T-type calcium channels.Thanks to their role in cellular excitability and calcium homeostasis, T-type calcium channels are involved in several physiological functions such as sleep or control of cardiac rythmicity and vascular tone. They are also involved in some diseases such as pain or epilepsy. The regulation of T-type calcium channels is still poorly understood and that is the challenge of this thesis.In a first part of the study, we caracterised the effect of some endogenous lipids on these channels, particularly the metabolites of arachidonic acid, and identified the 5,6-EET as a new blocker of T-type channels. We then evaluated the existence of a binding site of lipoamino acids on T-type channels using binding experiments made with a specific inhibitor of these channels, the TTA-A1 molecule.In a second part, we studied the regulation of T-type channels by calcium. We showed that a calcium entry through T-type channels or through P2X4 and NMDA receptors activation induced an inhibition of T-type current. Calcium would activate a phosphatase which would trigger a shift of the steady-state inactivation curve toward negative potentials, reducing the availability of channels. This phenomenon of current inhibition due to calcium entry may be a feedback mechanism limiting calcium entry in the cell to avoid toxicity due to a too high intracellular calcium concentration. After this inhibition, when calcium entry is stopped, the current increases. This increase seems to be due to the intervention of the Pak protein kinase which would make T-type channels available again.In conclusion, we studied and caracterised tow new mechanisms of T-type channels regulation: a modulation by lipids, and a modulation by calcium and the Pak protein kinase

[EN] ABSTRACT Abscisic acid (ABA) signaling plays a critical role in regulating root growth and root system architecture. ABA-mediated growth promotion and root tropic response under water stress are key responses for plant survival under limiting water conditions. In this work, we have explored the role of Arabidopsis (Arabidopsis thaliana) PYR/PYL/RCAR receptors (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS) for root ABA signaling. As a result, we discovered that PYL8 plays a nonredundant role for the regulation of root ABA sensitivity. Unexpectedly, given the multigenic nature and partial functional redundancy observed in the PYR/PYL family, the single pyl8 mutant showed reduced sensitivity to ABA-mediated root growth inhibition. This effect was due to the lack of PYL8-mediated inhibition of several clade A phosphatases type 2C (PP2Cs), since PYL8 interacted in vivo with at least five PP2Cs, namely HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 as revealed by tandem affinity purification and mass spectrometry proteomic approaches. Membrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calciumdependent interactions of PYR/PYL/RCAR ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA-independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant-specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL/RCAR receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL/RCAR function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, and CAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL/RCAR-interacting partners that mediate a transient Ca2+-dependent interaction with phospholipid vesicles, which affects PYR/PYL/RCAR subcellular localization and positively regulates ABA signaling.
[ES] RESUMEN La señalización por la hormona vegetal ácido abscísico (ABA) desempeña un papel crítico en la regulación del crecimiento de la raíz y en la arquitectura del sistema radical. La promoción de crecimiento de la raíz en condiciones de estrés hídrico mediada por ABA es clave para la supervivencia de las plantas bajo condiciones limitantes de agua. En este trabajo, hemos explorado el papel de los receptores PYR/PYL/RCAR (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/ REGULATORY COMPONENTS OF ABA RECEPTORS) de Arabidopsis (Arabidopsis thaliana) en la ruta de señalización de ABA en raíz. Así, hemos descubierto que el receptor de ABA PYL8 juega un papel no redundante en la regulación de la percepción de ABA en raíz. Inesperadamente, dada la naturaleza multigénica y la redundancia funcional parcial observada en la familia PYR/PYL/RCAR, el mutante pyl8 fue el único mutante sencillo de pérdida de función de los receptores PYR/PYL/RCAR que mostraba una sensibilidad reducida a la inhibición del crecimiento mediada por ABA en raíz. Este efecto se debe a la falta de inhibición mediada por PYL8 de varias fosfatasas del grupo A tipo 2C (PP2Cs), ya que PYL8 es capaz de interactuar in vivo con al menos cinco PP2Cs, denominadas HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 según lo han revelado la purificación por afinidad en tándem (TAP por sus siglas en inglés) y estudios proteómicos de espectrometría de masas. La transducción de la señal del ABA localizada en la membrana plasmática celular juega un papel crucial en los pasos iniciales de la señalización de la fitohormona, pero los mecanismos moleculares que unen los componentes básicos de la señalización y la membrana plasmática no están claros. Estudiando las interacciones de los receptores del ABA PYR/PYL/RCAR con la membrana plasmática hemos encontrado que éstos pueden interaccionar transitoriamente con ella de forma dependiente de calcio gracias a una familia de proteínas con dominios C2 relacionadas con la ruta de señalización de ABA (denominadas C2-domain ABA-related (CAR) proteins). Específicamente, se encontró que PYL4 interacciona de manera independiente de ABA con CAR1 tanto en la membrana plasmática como en el núcleo de las células vegetales. La proteína CAR1 pertenece a una familia multigénica constituida por 10 miembros en Arabidopsis thaliana, desde CAR1 hasta CAR10, y que solo se encuentra en plantas. Los ensayos de complementación bi-molecular de fluorescencia y de co-immunoprecipitación confirmaron la interacción en células vegetales tanto de PYL4-CAR1 como de otras parejas de PYR/PYL-CAR. La cristalización de la proteína CAR4 reveló que, además de un dominio C2 clásico de unión a lípidos dependiente de calcio, las proteínas de la familia CAR presentan un dominio específico que probablemente es responsable de la interacción con los receptores PYR/PYL/RCAR y de su posterior reclutamiento a las vesículas de fosfolípidos. Esta interacción es relevante para la función de los receptores PYR/PYL/RCAR en la señalización del ABA, ya que diferentes mutantes triples car de pérdida de función, que tienen afectados los genes CAR1, CAR4, CAR5, y CAR9, demostraron una reducción de la sensibilidad al ABA en ensayos de establecimiento de plántula y crecimiento de la raíz. En resumen, hemos identificado nueva familia de proteínas que son capaces mediar las interacciones transitorias dependientes de Ca2+ con vesículas de fosfolípidos, lo que a su vez afecta localización de PYR/PYL/RCAR y regula positivamente la señalización de ABA.
[CAT] RESUM La senyalització per l'hormona vegetal àcid abcíssic (ABA) exerceix un paper crític en la regulació del creixement de l'arrel i també en l'arquitectura del sistema radical. La promoció del creixement de l'arrel en condicions d'estrés hídric, regulada per ABA és clau per la supervivència de les plantes sota condicions limitants d'aigua. Amb aquest treball, hem investigat el paper dels receptors PYR/PYL/RCAR (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/ REGULATORY COMPONENTS OF ABA RECEPTORS) d'Arabidopsis (Arabidopsis thaliana) en el camí de senyalització d'ABA en arrel. Així, hem descobert que el receptor d'ABA PYL8 exerceix un paper no redundant en la regulació de la percepció d'ABA en arrel. Inesperadament, donada la naturalesa multigènica i la redundància funcional parcial que s'observa en la família PYR/PYL/RCAR, el mutant pyl8 va ser l'únic mutant senzill de pèrdua de funció dels receptors PYR/PYL/RCAR que mostrava una sensibilitat reduïda a la inhibició del creixement mitjançada per l'ABA en l'arrel. Doncs aquest efecte es deu a la falta d'inhibició regulada per PYL8 de diverses fosfatases del grup A tipus 2C (PP2Cs), ja que PYL8 té la capacitat d'interactuar in vivo almenys amb cinc PP2Cs, anomenades HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABAHYPERSENSITIVE GERMINATION3 segons ho han revelat per una banda la purificació per afinitat en tàndem (TAP són les seues sigles en anglés) i per altra banda, estudis proteòmics d'espectrometria de masses. Pel que fa a la transducció del senyal del l'ABA, la qual es localitza en la membrana plasmàtica cel¿lular, juga un paper molt important en els primers instants de la senyalització de la fitohormona, no obstant això els mecanismes moleculars que uneixen els components bàsics d'aquesta senyalització amb la membrana plasmàtica, no es troben del tot clars. Per tant, s'han estudiat les interaccions que tenen els receptors del ABA PYR/PYL/RCAR amb la membrana plasmàtica, i hem trobat que aquests tenen la capacitat d'interaccionar transitòriament amb la membrana de forma dependent al calci, gràcies a una família de proteïnes amb domini C2, les quals es troben relacionades amb la ruta de senyalització d'ABA(anomenades C2domain ABArelated (CAR) proteins).Específicament, es va trobar que PYL4 interacciona d'una manera independent al ABA amb CAR1, tant en la membrana plasmàtica, com en el nucli de les cèl¿lules vegetals. La proteïna CAR1 pertany a la família multigènica constituïda per 10 components en Arabidopsis thaliana, des de CAR1 fins CAR10, que tan sols es troba en plantes. Els assajos de complementació bimolecular de fluorescència i de co-immunoprecipitació, van confirmar la interacció en cèl¿lules vegetals, tant de PYL4CAR1 com d'altres parelles de PYR/PYL-CAR. La cristal¿lització de la proteïna CAR4 va revelar que, a més d'un domini C2 clàssic de unió a lípids dependent del calci, les proteïnes de la família CAR presenten un domini PYR/PYL/RCAR, i del seu posterior reclutament a les vesícules fosfolipídiques. Doncs, aquesta interacció és rellevant en la funció dels receptors PYR/PYL/RCAR, ja que participa en la senyalització del l'ABA. Aquesta interacció es clau per a la funció dels receptors, ja que diferents mutants triples car de pèrdua de funció, els quals posseïxen afectats els gens CAR1, CAR4, CAR5 i CAR9, van mostrar una reducció de la sensibilitat a l'ABA en assajos d'establiment de plàntula i creixement de l'arrel. En conclusió, hem identificat una nova família de proteïnes amb la capacitat d'organitzar les interaccions transitòries dependents del calci amb vesícules de fosfolípids, fet que al seu torn afecta la localització de PYR/PYL/RCAR i regula positivament la senyalització d'ABA.
Rodríguez Solovey, LN. (2015). IDENTIFICATION OF TARGETS AND AUXILIARY PROTEINS OF PYR/PYL/RCAR ABA RECEPTORS: PROTEIN PHOSPHATASES TYPE 2C (PP2Cs) AND C2-DOMAIN ABA-RELATED PROTEINS (CARs) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58862
TESIS

Dans ce travail, nous démontrons que STIM1, un senseur calcique activé par la déplétion du Ca2+ intracellulaire du réticulum endoplasmique, est indispensable pour la signalisation calcique et la préférence oro-sensorielle du gras. Nous observons que l'acide linoléique (LA), en activant les phospholipases A2 via CD36, produit de l'acide arachidonique (AA) et de la lyso-phosphatidylcholine (lyso-PC). Cette activation déclenche un influx calcique dans les cellules CD36-positives, et induit la production du facteur CIF (Ca2+ Influx Factor). CIF, AA et lyso-PC exercent différentes actions sur l'ouverture des canaux SOC (Stored Operated Calcium Channel) constitués de protéines Orai et contrôlés par STIM1. Par ailleurs, les souris au phénotype Stim1-/- perdent la préférence spontanée pour les lipides et la libération de la sérotonine à partir des cellules gustatives dans le milieu extracellulaire chez les animaux sauvages. Nous demontrons aussi que la signalisation calcique médiée via CD36 est doublement modulée lors de l'obésité. L'augmentation de la [Ca2+]i dans les cellules gustatives observée chez le Psammomys obesus, un modèle d'obésité nutritionelle, est fortement diminuée chez les souris rendues obèses par un regime hyperlipidique. Nous avons constaté également que l'interaction de LA avec le CD36 induit l'activation des MAP Kinases de la voie MEK1/2/ERK1/2/Elk-1 qui est non seulement à l'origine de l'activation des aires cérébrales telles que le NTS, le noyau arqué, l'hippocampe mais aussi indispensable pour la préférence spontanée pour les lipides alimentaires. Nos résultats suggèrent pour la prémière fois, que la voie ERK1/2 des MAPK et la signalisation calcique lipidique controlée par STIM1 sont impliquées dans la perception oro-gustative des lipides

碩士
國立臺灣科技大學
電子工程系
107
Lipid contamination from the intense subcutaneous lipid signal that produces Gibbs ringing artifacts may complicate the quantitation of metabolites in the Magnetic Resonance Spectroscopic Image. In 2D MRSI, lipid suppression is usually achieved by placing 8 outer volume saturation bands (OVS) around the brain with subcutaneous lipids or use inversion recovery to eliminate fat signals. In this study, post-processing method based on spatial signal projection (SSP) was used to achieve the effect of suppressing lipid signal. SSP extracts signal components of lipids and removes them to achieve effective lipid suppression. In this experiment, the effect of lipid suppressions was tested in brain phantoms. Data of brain phantom were collected by echo planar spectroscopic imaging (EPSI), and the concentration of metabolites after SSP lipid suppression was compared with the known concentration of brain phantom. The feasibility of SSP method for lipid suppression was demonstrated.

碩士
國立陽明大學
生理學研究所
91
Previous works indicated that Bacterial lipoplysaccharide (LPS) and it's toxically center, lipid A (LA), stimulated NF-κB mRNA expression and NF-κB protein activation, but not the proliferation in T cells. The increasing in intracellular calcium was through phosphoinositide 3-kinase (PI3K) pathway by Lipid A, whereas, the increases in intracellular pH (pHi) was through protein kinase C (PKC)/phospholipase D (PLD) pathway in T cells. Therefore, the aim of this study was to investigate whether gene expression by LPS or LA were regulated by differential signals stimulated in human peripheral T cells. The changes in intracellular calcium ([Ca2+]i) were measured by fluorescent dye, Fura-2, and the mRNA expression in Toll-like receptors (TLRs, TLR2 and TLR4), NF-κB and PLD were measured by reverse transcriptional-polymerase chain reaction (RT-PCR). The T cell proliferation was determined by [3H]-thymidine incorporation. The appearance of cell marker CD40L、TLR2、TLR4 on T cells were analyzed by flow cytometry. Inhibitors of PI3K, LY294002 and Wortmanin, inhibitor of PKCθ, Rottlerin, and inhibitor of protein tyrosine kinase (PTK), Genistein were used to explore the alternation on signals and the expression by lipid A. The results indicate that 1) The Ca2+ influx, but not intracellular calcium release by LA and was suppressed by Wortmanin and LY294002; 2) The mRNA expression on PLD、TLR4 was induced by LPS at 30 min and reached a peak at 1h. In addition, the mRNA expression on NF-κB and TLR2 was induced by LPS and reached a peak at 1 h. In contrast, LA did not stimulate PLD and TLR2 expression but increased the expression of NF-κB at 1h and reached a peak at 4 h; 3) The mRNA expression on NF-κB by LA was suppressed by pretreate cells with EGTA and Wortmanin, however, the expression on TLR4 was not affected; 4) Both LPS and LA did not affect the expression of cell surface protein marker CD40L. LPS increased the expression of cell surface protein markers, TLR2 and TLR4. Whereas, LA increased the expression of TLR2 but not TLR4; 5) Combinations of LA and PMA did not alter [3H]-thymidine incorporation into T cells. In summary, LPS could induce the mRNA expression on PLD, NF-κB, TLR2 and TLR4 as well as the cell surface protein on TLR2 and TLR4. In contrast, LA only increased mRNA expression of NF-κB and TLR4 as well as the cell surface protein on TLR2. In conclusion, the mRNA expression on PLD and TLR2 was the only differential signal that found by LPS and LA, thus, LPS had better enhancement than LA on T cell natural immunity.

The epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase integral in regulating cell growth, survival, and migration. EGFR signaling, which is dependent on localization of the receptor within lipid rafts, is often hijacked during colon tumorigenesis. Previous work has found that docosahexaenoic acid (DHA) is protective against colon cancer. This fatty acid is proposed to function in part by perturbing lipid rafts and thereby altering cell signaling. The overall objective of this work was to determine whether DHA alters EGFR function and signaling. We assessed EGFR localization and ligand-induced phosphorylation in YAMC cells treated with fatty acids. We found that DHA reduced the localization of EGFR to lipid rafts. Concomitant with altering receptor localization, DHA was found to increase EGFR phosphorylation. However, DHA paradoxically suppressed EGFR signal transduction. We found that DHA uniquely altered EGFR activity, and other long chain polyunsaturated fatty acid did not exert the same effect. We additionally observed similar effects on EGFR activation and signaling by feeding mice a diet enriched in fish oil (high in DHA), and this was attendant with reduced colon tumorigenesis. We next probed the mechanism by which DHA enhances EGFR phosphorylation. We found that DHA facilitates receptor dimerization to increase phosphorylation. We additionally identified Ras activation as the site of perturbation of signal transduction. DHA suppressed signal transduction by both changing the localization of EGFR within the plasma membrane and increasing receptor endocytosis and degradation. Lastly, we extended our observations into a wounding model. Although DHA uniquely altered ligand-stimulated EGFR activity, both DHA and EPA altered EGFR transactivation and signaling upon injury. This culminated in reduced wound healing in DHA and EPA treated cells. In an animal model, we found that diets enriched in either DHA or EPA altered EGFR signaling in the colonocytes of wounded animals. Overall, we found that DHA modifies EGFR signaling, which can be beneficial or detrimental for health depending on the disease state of an individual. These data help elucidate a mechanism by which DHA protects against colon cancer, as well as indicating a potential downside of n-3 PUFA therapy.

Indiana University-Purdue University Indianapolis (IUPUI)
Carbon nanomaterials are widely produced and used in industry, medicine and scientific research. To examine the impact of exposure to nanoparticles on human health, the human airway epithelial cell line, Calu-3, was used to evaluate changes in the cellular proteome that could account for alterations in cellular function of airway epithelia after 24 h exposure to 10 μg/mL and 100 ng/mL of two common carbon nanoparticles, singleand multi-wall carbon nanotubes (SWCNT, MWCNT). After exposure to the nanoparticles, label-free quantitative mass spectrometry (LFQMS) was used to study differential protein expression. Ingenuity Pathway Analysis (IPA) was used to conduct a bioinformatics analysis of proteins identified by LFQMS. Interestingly, after exposure to a high concentration (10 μg/mL; 0.4 μg/cm2) of MWCNT or SWCNT, only 8 and 13 proteins, respectively, exhibited changes in abundance. In contrast, the abundance of hundreds of proteins was altered in response to a low concentration (100 ng/mL; 4 ng/cm2) of either CNT. Of the 281 and 282 proteins that were significantly altered in response to MWCNT or SWCNT, respectively, 231 proteins were the same. Bioinformatic analyses found that the proteins common to both kinds of nanotubes are associated with the cellular functions of cell death and survival, cell-to-cell signaling and interaction, cellular assembly and organization, cellular growth and proliferation, infectious disease, molecular transport and protein synthesis. The decrease in expression of the majority proteins suggests a general stress response to protect cells. The STRING database was used to analyze the various functional protein networks. Interestingly, some proteins like cadherin 1 (CDH1), signal transducer and activator of transcription 1 (STAT1), junction plakoglobin (JUP), and apoptosis-associated speck-like protein containing a CARD (PYCARD), appear in several functional categories and tend to be in the center of the networks. This central positioning suggests they may play important roles in multiple cellular functions and activities that are altered in response to carbon nanotube exposure. To examine the effect of nanotubes on the plasma membrane, we investigated the interaction of short purified MWCNT with model lipid membranes using a planar bilayer workstation. Bilayer lipid membranes were synthesized using neutral 1, 2-diphytanoylsn-glycero-3-phosphocholine (DPhPC) in 1 M KCl. The ion channel model protein, Gramicidin A (gA), was incorporated into the bilayers and used to measure the effect of MWCNT on ion transport. The opening and closing of ion channels, amplitude of current, and open probability and lifetime of ion channels were measured and analyzed by Clampfit. The presence of an intermediate concentration of MWCNT (2 μg/ml) could be related to a statistically significant decrease of the open probability and lifetime of gA channels. The proteomic studies revealed changes in response to CNT exposure. An analysis of the changes using multiple databases revealed alterations in pathways, which were consistent with the physiological changes that were observed in cultured cells exposed to very low concentrations of CNT. The physiological changes included the break down of the barrier function and the inhibition of the mucocillary clearance, both of which could increase the risk of CNT’s toxicity to human health. The biophysical studies indicate MWCNTs have an effect on single channel kinetics of Gramicidin A model cation channel. These changes are consistent with the inhibitory effect of nanoparticles on hormone stimulated transepithelial ion flux, but additional experiments will be necessary to substantiate this correlation.

Phospholipase Cβ (PLCβ) cleaves phosphatidylinositol-4,5-bisphosphate (PIP₂) into the second messengers inositol-1,4,5-triphosphate (IP₃) and diacylglycerol (DAG). IP₃ increases intracellular Ca²⁺, while DAG remains in the membrane, and together with increased Ca²⁺, activates protein kinase C (PKC). PLCβ has low basal activity but is activated following stimulation of G_i- and G_q-coupled receptors through direct interactions with Gα_q and Gβγ. PLCβ is essential for normal cardiomyocyte and vascular smooth muscle function and regulates cell proliferation, survival, migration, and differentiation. However, increased PLCβ activity and expression results in arrhythmias, hypertrophy, and heart failure. PLCβ must interact with the cell membrane for its activity. While heterotrimeric G proteins stimulate PLCβ, they are insufficient for full activation, suggesting the membrane itself contributes to increased lipid hydrolysis, potentially via interfacial activation. However, how the composition of the membrane and its resulting properties, such as surface charge, contribute to adsorption and interfacial activation is not well-established. Furthermore, whether or how interfacial activation also impacts other regulatory elements in PLCβ and Gα_q-dependent activation is unknown. Using an innovative combination of atomic force microscopy on compressed lipid monolayers and biochemical assays, we are beginning to understand how the membrane itself, PLCβ autoinhibitory elements and Gα_q regulate PLCβ activation. These studies provide the first structure-based approach to understanding how the cell membrane regulates the activity of this essential effector enzyme.