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The Wingless (Wnt) signaling pathway is one of the critical developmental pathways for control of cell differentiation, proliferation, and cell growth. The DEP domain, located on the C-terminus of Dishevelled (Dvl), plays a role in cytoplasm-membrane association, which branches the canonical and non-canonical Wnt signaling pathway within the cell. It has been suggested that the DEP domain requires the recruitment of ionic lipids, such as phosphatidic acid, to regulate its localization to the plasma membrane and association to the frizzle receptor. However, the physical mechanism for DEP association to the plasma membrane is still unknown. We show that mDvl2-DEP interacts with phosphatidic acid at a distinct patch on the surface formed by a positively charged surface area by NMR spectroscopy. The binding of this interaction was also found at physiologically relevant concentration using fluorescence spectroscopy. We also determined that the interaction is pH-dependent and regulated through a 'histidine switch' mechanism at His464 and His465 where there is increased association of mDvl2-DEP to the plasma membrane at higher pH values (7.5). This association is based on tertiary structure conformational changes with rearrangement of the loop regions by a change in local pH, not its interaction with phosphatidic acid. Overall, our work will contribute to elucidate how cells regulate their developmental pathways through localized molecular interactions. / Master of Science

Emerging evidence suggests that an important mechanism for the negative feedback control of insulin signalling involves the inhibition of tyrosine phosphorylation of IRS-1 by its prior serine/threonine (ser/thr) phosphorylation. IRS-1 ser/thr phosphorylation has been linked to the dissociation of IRS-1 from the insulin receptor and PI3K, and its degradation via a proteasome-dependent pathway. Studies in animal models have shown that increases in plasma free fatty acids (FFAs) are associated with reduced IRS-1-signalling, and so it has been postulated that elevated FFA cause insulin resistance by activating pathways that negatively regulate insulin action, including hyper-phosphorylation of ser/thr residues in IRS-1. We have shown that in the case of linoleate-induced insulin resistance in L6 rat skeletal muscle cells, the inhibition of IRS-1-dependent signalling arises via effects on both the phosphorylation status and degradation of IRS-1, which are mediated, in part, by IKKb. In addition, the reduction of IRS-1 mRNA levels allude to transcriptional effects of linoleate treatment that also contribute to the observed reduction in the total levels of this protein. PtdOH, particularly dilinoleoyl PtdOH, was found to be significantly increased in linoleate treated L6 cells, and sufficient to induce at least some of the effects on insulin-signalling that are observed upon linoleate treatment. It is unlikely, however, that IKKb and PtdOH are components of the same inhibitory pathway, since inhibiting IKKb activity did not alleviate the effects of PtdOH on IRS-1 tyrosine (tyr) phosphorylation. Moreover, although an integral component of the mechanism by which linoleate induces insulin-resistance in L6 cells, it appears that restoring IRS-1 function in linoleate treated cells is not sufficient to reverse insulin resistance. Hence, we hypothesise that linoleate induces multiple inhibitory pathways in L6 cells, with at last two of these involving IKKb- and PtdOH-dependent inhibition of IRS-1 signalling, which act in parallel to reduce glucose disposal and cause insulin resistance in this model.

A family of diacylglycerol kinases (DGK) phosphorylates the substrate diacylglycerol (DAG) to generate phosphatidic acid (PA) . Both molecules, DAG and PA, are involved in signal transduction pathways. In the model plant Arabidopsis thaliana, seven candidate genes (named AtDGK1 to AtDGK7) code for putative DGK isoforms. Here I report the molecular cloning and characterization of AtDGK7. Biochemical, molecular and physiological experiments of AtDGK7 and their corresponding enzyme are analyzed. Information from Genevestigator says that AtDGK7 gene is expressed in seedlings and adult Arabidopsis plants, especially in flowers. The AtDGK7 gene encodes the smallest functional DGK predicted in higher plants; but also, has an alternative coding sequence containing an extended AtDGK7 open reading frame, confirmed by PCR and submitted to the GenBank database (under the accession number DQ350135). The new cDNA has an extension of 439 nucleotides coding for 118 additional amino acids The former AtDGK7 enzyme has a predicted molecular mass of ~41 kDa and its activity is affected by pH and detergents. The DGK inhibitor R59022 also affects AtDGK7 activity, although at higher concentrations (i.e. IC50 ~380 µM). The AtDGK7 enzyme also shows a Michaelis-Menten type saturation curve for 1,2-DOG. Calculated Km and Vmax were 36 µM 1,2-DOG and 0.18 pmol PA min-1 mg of protein-1, respectively, under the assay conditions. Former protein AtDGK7 are able to phosphorylate different DAG analogs that are typically found in plants. The new deduced AtDGK7 protein harbors the catalytic DGKc and accessory domains DGKa, instead the truncated one as the former AtDGK7 protein (Gomez-Merino et al., 2005). / Wachstum und Entwicklung sind die Kennzeichen lebender Systeme. Diese Prozesse unterliegen einer strengen Regulation im Organismus. Diacylglycerol (DAG) und Phosphatidsäure (PA) sind wesentliche Elemente in der Signalübertragung in Organismen. In Säugetieren kann DAG auf drei verschiedenen Wegen metabolisiert werden, die Entstehung von PA durch Phosphorylierung der freien Hydroxyl-Gruppe von DAG ist jedoch der am häufigsten vorkommende Stoffwechselweg. Die enzymatische Umsetzung dieser Reaktion wird von der Familie der Diacylglycerol-Kinasen (DGKs) katalysiert. Molekulare und biochemische Untersuchungen konnten die Anwesenheit von DGKs in Drosophila melanogaster, Arabidopsis thaliana und jüngst auch in Dictyostelium discoideum zeigen. In der vorliegenden Arbeit wird die Klonierung und Charakterisierung von AtDGK7 aus Arabidopsis thaliana präsentiert, einem Vertreter des pflanzlichen DGK-Clusters II. Das Transkript von AtDGK7 findet sich in der gesamten Pflanze, jedoch sind die Transkriptmengen in Blüten und jungem Gewebe stark erhöht. Rekombinant hergestelltes AtDGK7 ist katalytisch aktiv und akzeptiert DAG-ähnliche Moleküle mit mindestens einer ungesättigten Fettsäure als bevorzugtes Substrat. AtDGK2, ein weiteres Mitglied der DGK-Familie, und AtDGK7 metabolisieren Substrate, welche in Pflanzen physiologisch relevant sind. Das als DGK-Inhibitor beschriebene Molekül 6-{2-{4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl}ethyl}-7-methyl-5H-thiazolo(3,2-a)pyrimidine-5-one (R59022) inhibiert bei Konzentrationen von 50-100 µM rekombinant hergestelltes AtDGK2 in vitro. In ähnlichen Konzentrationen eingesetzt modifiziert R59022 das Wurzelwachstum. Dies weist darauf hin, dass DGKs in Entwicklungsprozessen eine Rolle spielen. In in vitro Experimenten wurde AtDGK7 von R59022 allerdings erst in Konzentrationen über 100 µM inhibiert. Ferner wird in der vorliegenden Arbeit die erfolgreiche Klonierung einer cDNA beschrieben, die für AtDGK7 aus A. thaliana kodiert und welche im Vergleich zu der bereits bekannten cDNA um 439 bp länger ist. Expressionsanalysen mit Hilfe eines Promotor-ß-glucuronidase (GUS) Fusions-Produktes zeigten die Aktivität von AtDGK7 in vielen Geweben, vor allem aber in Schließzellen, im Konnektiv-Gewebe der Antheren, sowie besonders in den Spitzen der Seitenwurzeln. Physiologische Untersuchungen unter abiotischem Stress (Verwendung verschiedener Konzentrationen von Stickstoff, Saccharose, Auxin und Inhibitoren von Auxin-Transportern) wurden mit AtDGK7 T-DNA-Insertionslinien sowie mit den Promotor-GUS-Linien durchgeführt. AtDGK7 T-DNA-Insertionslinien zeigten eine starke Inhibierung des Seitenwurzel-Wachstums unter limitierenden Stickstoff- und/oder Saccharose-Konzentrationen. In einigen der T-DNA-Insertionslinien inhibierte die Zugabe eines Inhibitors für Auxin-Transport (TIBA; 2,3,5-triiodobenzoic acid) die Bildung von Haupt- und Seitenwurzeln fast vollständig. Die Inhibition des Wurzelwachstums in den T-DNA-Insertionslinien konnte teilweise durch die Zugabe von 50nM NAA (α-naphtalene acetic acid) revertiert werden. Aus den vorliegenden Ergebnissen wird die Hypothese abgeleitet, dass AtDGK7 im Zusammenspiel mit Auxin in Signaltransduktionsprozessen eine Rolle spielt, welche das Wachstum und die Entwicklung in Pflanzen regulieren.

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

Phosphatidic Acid (PA) is a natural phospholipid compound derived from lecithin which is commonly found in egg yolk, grains, fish, soybeans, peanuts and yeast. It has been suggested that PA is involved in several intracellular processes associated with muscle hypertrophy. Specifically, PA has been reported to activate protein synthesis through the mammalian target of rapamycin (mTOR) signaling pathway and thereby may enhance the anabolic effects of resistance training. To our knowledge, no one has examined the effect of PA supplementation in humans while undergoing a progressive resistance training program. To examine the effect of PA supplementation on lean soft tissue mass (LM) and strength after 8 weeks of resistance training. Fourteen resistance-trained men (mean ± SD; age 22.7 ± 3.3 yrs; height: 1.78 ± 0.10m; weight: 89.3 ± 16.3 kg) volunteered to participate in this randomized, double-blind, placebocontrolled, repeated measures study. The participants were assigned to a PA group (750mg/day; Mediator®, ChemiNutra, MN, n=7) or placebo group (PL; rice flower; n=7), delivered in capsule form that was identical in size, shape and color. Participants were tested for 1RM strength in the bench press (BP) and squat (SQ) exercise. LM was measured using dual-energy X-ray absorptiometry. After base line testing, the participants began supplementing PA or PLfor 8 weeks during a progressive resistance training program intended for muscular hypertrophy. Data was analyzed using magnitude-based inferences on mean changes for BP, SQ and LM. Furthermore, the magnitudes of the interrelationships between changes in total training volume and LM were interpreted using Pearson correlation coefficients, which had uncertainty (90% confidence limits) of approximately +0.25. iv In the PA group, the relationship between changes in training volume and LM was large(r=0.69, +0.27; 90%CL), however, in the PL group the relationship was small (r=0.21, +0.44; 90%CL). PA supplementation was determined to be likely beneficial at improving SQ and LM over PL by 26% and 64%, respectively. The strong relationship between changes in total training volume and LM in the PA group suggest that greater training volume most likely lead to the greater changes in LM, however, no such relationship was found with PL group. For the BP data, the PA group resulted in a 42% greater increase in strength over PL, although the effect was considered unclear. While more research is needed to elucidate mechanism of action; the current findings suggest that in experienced resistance trained men supplementing 750mg PA per day for 8 weeks may likely benefit greater changes in muscle mass and strength compared with resistance training only.

No description available.

Protein–lipid binding interactions play crucial roles in various physiological and pathological processes, making it very important to study these interactions at the molecular level. However, investigation of these interactions is complicated by several issues, including the inherent complexity of membranes as well as the diverse mechanisms by which proteins interact with the membrane surfaces. As a result, many of these interactions remain poorly characterized. Synthetic probes are useful tools employed for studying protein–lipid binding interactions. This thesis will detail the design and synthesis of metabolically stabilized analogues of various signaling lipids, which mimic the natural species and are not easily modified by enzymes present in biological systems. A modular approach is employed for synthesizing these lipid probes, giving access to a wide range of derivatized lipid probes that can then be used for several studies. Although a wide variety of metabolically stabilized lipid analogues have been synthesized, their activity has not yet been characterized and quantified in detail. So, there is a great need to synthesize biologically active phosphorothioate and phosphonate analogues of various signaling lipids in order to properly characterize and compare the binding affinities and activity of these analogues. Synthesis of metabolically stabilized lipid analogue would take us one step closer towards understanding protein–lipid interactions in biological systems and in trying to find answers to the myriad of questions pertaining to these systems.

L’acide phosphatidique (PA) est un lipide simple qui peut exister sous différentes formes. A partir des sondes que j’ai préparé en se basant sur des domaines de liaison au PA : PDE4A1, Spo20p, et OpiQ2, j’ai pu étudier la localisation subcellulaire du PA dans les cellules PC12 et les macrophages RAW264.7. Ces sondes lient différents formes de PA dans les membranes de différents compartiments subcellulaires. De plus, j’ai pu montrer qu’il y a une néosynthèse de PA et de certaines espèces de PA mono- ou bi-insaturé à la membrane plasmique lors de la stimulation de l’exocytose. Nous avons ainsi observé que la PLD1 semble être la source principale de PA dans les glandes surrénales. D’autre part, mes travaux indiquent une augmentation du niveau global de PA à la membrane plasmique et une diminution importante du PA au niveau du RE dans les macrophages après stimulation de la phagocytose frustrée. Ce qui pourrait ainsi valider le concept d’une fusion d’une partie de la membrane du RE avec la membrane plasmique lors de la phagocytose. / The phosphatidic acid (PA) is a simple lipid which may exist in various forms. I have generated probes based on PA binding domains: PDE4A1, Spo20p and OpiQ2 to study the subcellular localization of PA in PC12 cells and RAW264.7 macrophages. These probes bind different form of PA in different subcellular compartments. In addition, I show that PA and certain species mono- or bi-unsaturated of PA are synthesized at the plasma membrane upon stimulation of exocytosis. We observed that the PLD1 seems to be the main source of PA in the adrenal glands. On the other hand, my research indicates an increase in the level of PA at the plasma membrane and a significant decrease in the ER in macrophages after stimulation of phagocytosis frustrated. Thus these results could validate the concept of a fusion of a portion of the ER membrane with the plasma membrane during phagocytosis.

No description available.