Relationships between EEA1 binding partners and their role in endosome fusion

Homotypic fusion between early endosomes requires the phosphatidylinositol 3-phosphate (PI3P)-binding protein, Early Endosomal Autoantigen 1 (EEA1). We have investigated the role of other proteins that interact with EEA1 in the fusion of early endosomes derived from Baby Hamster Kidney (BHK) cells. We confirm a requirement for syntaxin 13, but additionally show that the assay is equally sensitive to reagents specifically targeted against syntaxin 6. Binding of EEA1 to immobilised GST-syntaxin 6 and 13 was directly compared; only syntaxin 6 formed a stable complex with EEA1. Early endosome fusion requires the release of intravesicular calcium, and calmodulin plays a vital role in the fusion pathway, as judged by sensitivity to antagonists. We demonstrate that both EEA1 and syntaxin 13 interact with calmodulin. In the case of EEA1, binding to calmodulin requires an IQ domain, which is adjacent to a C-terminal FYVE domain that specifically binds to PI3P. We have assessed the influence of protein binding partners on EEA1 interaction with PI3P and find that both calmodulin and rab5-GTP are antagonistic to PI3P binding, whilst syntaxins 6 and 13 have no effect. These studies reveal a complex network of interactions between the proteins required for endosome fusion.

The sarcolemmal calcium pump inhibits the calcineurin/nuclear factor of activated T-cell pathway via interaction with the calcineurin A catalytic subunit

The calcineurin/nuclear factor of activated T-cell (NFAT) pathway represents a crucial transducer of cellular function. There is increasing evidence placing the sarcolemmal calcium pump, or plasma membrane calcium/calmodulin ATPase pump (PMCA), as a potential modulator of signal transduction pathways. We demonstrate a novel interaction between PMCA and the calcium/calmodulin-dependent phosphatase, calcineurin, in mammalian cells. The interaction domains were located to the catalytic domain of PMCA4b and the catalytic domain of the calcineurin A subunit. Endogenous calcineurin activity, assessed by measuring the transcriptional activity of its best characterized substrate, NFAT, was significantly inhibited by 60% in the presence of ectopic PMCA4b. This inhibition was notably reversed by the co-expression of the PMCA4b interaction domain, demonstrating the functional significance of this interaction. PMCA4b was, however, unable to confer its inhibitory effect in the presence of a calcium/calmodulin-independent constitutively active mutant calcineurin A suggesting a calcium/calmodulin-dependent mechanism. The modulatory function of PMCA4b is further supported by the observation that endogenous calcineurin moves from the cytoplasm to the plasma membrane when PMCA4b is overexpressed. We suggest recruitment by PMCA4b of calcineurin to a low calcium environment as a possible explanation for these findings. In summary, our results offer strong evidence for a novel functional interaction between PMCA and calcineurin, suggesting a role for PMCA as a negative modulator of calcineurin-mediated signaling pathways in mammalian cells. This study reinforces the emerging role of PMCA as a molecular organizer and regulator of signaling transduction pathways.

Novel functional interaction between the plasma membrane Ca2+ pump 4b and the proapoptotic tumor suppressor Ras-associated factor 1 (RASSF1)

Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling.

Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in the regulation of metabolic activity in cancer and immune cells, and affects whole-body metabolism by regulating ghrelin-signalling in the hypothalamus. This has led to efforts to develop specific CaMKK2 inhibitors, and STO-609 is the standardly used CaMKK2 inhibitor to date. We have developed a novel fluorescence-based assay by exploiting the intrinsic fluorescence properties of STO-609. Here, we report an in vitro binding constant of KD ∼17 nM between STO-609 and purified CaMKK2 or CaMKK2:Calmodulin complex. Whereas high concentrations of ATP were able to displace STO-609 from the kinase, GTP was unable to achieve this confirming the specificity of this association. Recent structural studies on the kinase domain of CaMKK2 had implicated a number of amino acids involved in the binding of STO-609. Our fluorescent assay enabled us to confirm that Phe(267) is critically important for this association since mutation of this residue to a glycine abolished the binding of STO-609. An ATP replacement assay, as well as the mutation of the 'gatekeeper' amino acid Phe(267)Gly, confirmed the specificity of the assay and once more confirmed the strong binding of STO-609 to the kinase. In further characterising the purified kinase and kinase-calmodulin complex we identified a number of phosphorylation sites some of which corroborated previously reported CaMKK2 phosphorylation and some of which, particularly in the activation segment, were novel phosphorylation events. In conclusion, the intrinsic fluorescent properties of STO-609 provide a great opportunity to utilise this drug to label the ATP-binding pocket and probe the impact of mutations and other regulatory modifications and interactions on the pocket. It is however clear that the number of phosphorylation sites on CaMKK2 will pose a challenge in studying the impact of phosphorylation on the pocket unless the field can develop approaches to control the spectrum of modifications that occur during recombinant protein expression in E. coli.

Evolution of the extracellular matrix in deuterostomes and the influence of calcitropic hormones

Tese de dout., Biologia (Biologia Molecular), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010

Developmental switch in the kinase dependency of long-term potentiation depends on expression of GluA4 subunit-containing AMPA receptors

The AMPA-receptor subunit GluA4 is expressed transiently in CA1 pyramidal neurons at the time synaptic connectivity is forming, but its physiological significance is unknown. Here we show that GluA4 expression is sufficient to alter the signaling requirements of long-term potentiation (LTP) and can fully explain the switch in the LTP kinase dependency from PKA to Ca2(+)/calmodulin-dependent protein kinase II during synapse maturation. At immature synapses, activation of PKA leads to a robust potentiation of AMPA-receptor function via the mobilization of GluA4. Analysis of GluA4-deficient mice indicates that this mechanism is critical for neonatal PKA-dependent LTP. Furthermore, lentiviral expression of GluA4 in CA1 neurons conferred a PKA-dependent synaptic potentiation and LTP regardless of the developmental stage. Thus, GluA4 defines the signaling requirements for LTP and silent synapse activation during a critical period of synapse development.

Molecular epidemiology of Aspergillus collected from cystic fibrosis patients

Background - Aspergillus respiratory infection is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function and allergic disease. Methods - Fifty-three Aspergillus isolates recovered from CF patients were identified to species by Internal Transcribed Spacer Region (ITS), β-tubulin, and calmodulin sequencing. Results - Three species complexes (Terrei, Nigri, and Fumigati) were found. Identification to species level gave a single Aspergillus terreus sensu stricto, one Aspergillus niger sensu stricto and 51 Aspergillus fumigatus sensu stricto isolates. No cryptic species were found. Conclusions - To our knowledge, this is the first prospective study of Aspergillus species in CF using molecular methods. The paucity of non-A. fumigatus and of cryptic species of A. fumigatus suggests a special association of A. fumigatus sensu stricto with CF airways, indicating it likely displays unique characteristics making it suitable for chronic residence in that milieu. These findings could refine an epidemiologic and therapeutic approach geared to this pathogen.

The role of cyclic nucleotides in modulation of crayfish neuromuscular junctions by a neuropeptide /

DF2, a heptapeptide, is a member of the family of FMRFamide-like peptides and has been shown to increase the amount of transmitter released at neuromuscular junctions of the crayfish, Procambarus clarkit Recent evidence has shown that protein kinase C (PKC), calcium/calmodulin-dependent protein kinase II (CaMKII) and the cAMPdependent protein kinase (PKA) play a role in the neuromodulatory pathway of DF2. The involvement of these kinases led to the prediction that a G-protein-coupled receptor (GPCR) is activated by DF2 due to the role that each kinase plays in traditional GPCR pathways seen in other organisms and in other cells. G-proteins can also act on an enzyme that generates cyclic guanosine monophosphate (cGMP) which mediates its effects through a cGMP-dependent protein kinase (PKG). This thesis addresses the question of whether or not DF2's effects on synaptic transmission in crayfish are mediated by the cyclic nucleotides cAMP and cGMP. The effects of DF2 on synaptic transmission were examined using deep abdominal extensor muscles of the crayfish Procambarus clarkii. An identified motor neuron was stimulated, and excitatory post-synaptic potentials (EPSPs) were recorded in abdominal extensor muscle LI . A number of activators and inhibitors were used to determine whether or not cAMP, PKA, cGMP and PKG mediate the effect of this peptide. Chemicals that are known to activate PKA (Sp-cAMPS) and/or PKG (8-pCPTcGMP) mimic and potentiate DF2's effect by increasing EPSP amplitude. Inhibitors of either PKA (Rp-cAMPS) or PKG (Rp-8-pCPT-cGMPS) block a portion of the increase in EPSP amplitude induced by the peptide. When both kinase inhibitors are applied simultaneously, the entire effect of DF2 on EPSPs is blocked. The PKG inhibitor blocks the effects of a PKG activator but does not alter the effect of a PKA activator on EPSP amplitude. Thus, the PKG inhibitor appears to be relatively specific for PKG. A trend in the data suggests that the PKA inhibitor blocks a portion of the response elicited by the PKG activator. Thus, the PKA inhibitor may be less specific for PKA. Phosphodiesterase inhibitors, which are known to inhibit the breakdown of cAMP (IBMX) and/or cGMP (mdBAMQ), potentiate the effect of the peptide. These results support the hypothesis that cAMP and cGMP, acting through their respective protein kinase enzymes, mediate the ability of DFi to increase transmitter output.

Cytosolic calcium levels and stress induced y-amino butyrate synthesis in asparagus mesophyll cells

Numerous investigations have demonstrated large increases in y-amino butyrate (GABA) levels in response to a variety of stresses such as touch or cold shock (Wallace et ale 1984) Circumstantial evidence indicating a role of Ca2 + in these increases includes elevated Ca2+ levels in response to touch and cold shock (Knight et ale 1991), and the demonstration of a calmodulin binding domain on glutamate decarboxylase (GAD), the enzyme responsible for GABA synthesis (Baum et al 1993) In the present study the possible role of Ca2+ and calmodulin in stimulation of GAD and subsequent GABA accumulation was examined using asparagus mesophyll cells. Images of cells loaded with the Ca2+ indicator Fluo-3 revealed a rapid and transient increase in cytosolic Ca2+ in response to cold shock. GABA levels increased by 106% within 15 min. of cold shock. This increase was inhibited 70% by the calmodulin antagonist W7, and 42% by the Ca2+ channel blocker La3+.. Artificial elevation of intracellular Ca2+ by the Ca2+ionophore A23187 resulted in an 61% increase in GABA levels. Stimulation of GABA synthesis by ABA resulted in an 83% increase in GABA levels which was inhibited 55% by W7. These results support the hypothesis that cold shock stimulates Ca2+ entry into the cytosol of the cells which results in Ca2+/calmodulin mediated activation of GAD and consequent GABA synthesis.

GABA accumulation and the hypersensitive response in isolated mesophyll cells treated with the G protein activator mastoparan

GABA (y-amino butyric acid) is a non-protein amino acid synthesized through the a-decarboxylation of L-glutamate. This reaction is catalyzed by L-glutamate decarboxylase (EC 4.1.1.15), a cytosolic Ca2+/calmodulin-stimulated enzyme. The purpose of this study is to determine whether or not GABA accumulation is associated with the hypersensitive response of isolated Asparagus sprengeri mesophyll cells. The addition of 25 J.lM mastoparan, a G protein activator, to suspensions of isolated asparagus mesophyll cells significantly increased GABA synthesis and cell death. Cell death was assessed using Evan's blue dye and fluorescein diacetate tests for cell viability. In addition, mastoparan stimulated pH-dependent alkalinization of the external medium, and a rapid and large 02 consumption followed by a loss of photosynthetic activity. The rate of 02 consumption and the net decrease in 02 in the dark was enhanced by light. The inactive mastoparan analogue Mas17 was ineffective in stimulating GABA accumulation, medium alkalinization, 02 uptake and cell death. Accumulation of H202 in response tomastoparan was not detected, however, mastoparan caused the cell-dependent degradation of added H202. The pH dependence of mastoparan-stimulated alkalinization suggests cellular electrolyte leakage, while the consumption of 02 corresponds to the oxidative burst in which 02 at the cell surface is reduced to form various active oxygen species. The results are indicative of the "hypersensitive response" of plants to pathogen attack, namely, the death of cells in the locality of pathogen invasion. The data are compatible with a model in which mastoparan triggers G protein activity, subsequent intracellular signal transduction pathway/s, and the hypersensitive response. It is postulated that the physiological elicitation of the hypersensitive response involves G protein signal transduction. The synthesis of GABA during the hypersensitive response has not been documented previously; however the role/s of GABA synthesis in the hypersensitive response, if any, remain unclear.

Rapid [Gamma]-amino acid synthesis and the inhibition of the growth and development of oblique banded leaf-roller larvae

The hypothesis that rapid y-aminobutyric acid (GABA) accumulation is a plant defense against phytophagous insects was investigated. Simulation of mechanical damage resulting from phytophagous insect activity increased soybean (Glycine max L.) leaf GABA 10- to 25-fold within 1 to 4 min. Pulverizing leaf tissue resulted in a value of 2. 15 (±O. 11 SE) ~mol GABA per gram fresh weight. Increasing the GABA levels in a synthetic diet from 1.6 to 2.6 Jlffiol GABA per gram fresh weight reduced the growth rates, developmental rates, total biomass (50% reduction), and survival rates (30% reduction) of cultured Oblique banded leaf-roller (OBLR) (Choristonellra rosacealla Harris) larvae. In field experiments OBLR larvae were found predominantly on young terminal leaves which have a reduced capacity to produce GABA in response to mechanical damage. Glutamate decarboxylase (GAD) is a cytosolic enzyme which catalyses the decarboxylation of L-Glu to GABA. GAD is a calmodulin binding enzyme whose activity is stimulated dramatically by increased cytosolic H+ or Ca2 + ion concentrations. Phytophagous insect activity will disrupt the cellular compartmentation of H+ and Ca2 +, activate GAD and subsequent GABA accumulation. In animals GABA is a major inhibitory neurotransmitter. The possible mechanisms resulting in GABA inhibited growth and development of insects are discussed.

The influence of myosin regulatory light chain phosphorylation on the contractile performance of fatigued mammalian skeletal muscle

ABSTRACT The myosm regulatory light chain (RLC) of type II fibres is phosphorylated by Ca2+ -calmodulin dependent myosin light chain kinase (skMLCK) during muscular activation. The purpose of this study was to explore the effect of skMLCK gene ablation on the fatigability of mouse skeletal muscles during repetitive stimulation. The absence of myosin RLC phosphorylation in skMLCK knockout muscles attenuated contractile performance without a significant metabolic cost. Twitch force was potentiated to a greater extent in wildtype muscles until peak force had diminished to ~60% of baseline (37.2 ± 0.05% vs. 14.3 ± 0.02%). Despite no difference in peak force (Po) and shortening velocity (Vo), rate of force development (+dP/dt) and shortening-induced deactivation (SID) were almost two-fold greater in WT muscles. The present results demonstrate that myosin RLC phosphorylation may improve contractile performance during fatigue; providing a contractile advantage to working muscles and protecting against progressive fatigue.

Mode of action of a Drosophila FMRFamide in inducing muscle contraction

Drosophila melanogaster is a model system for examining the mechanisms of action of neuropeptides. DPKQDFMRFamide was previously shown to induce contractions in Drosophila body wall muscle fibres in a Ca(2+)-dependent manner. The present study examined the possible involvement of a G-protein-coupled receptor and second messengers in mediating this myotropic effect after removal of the central nervous system. DPKQDFMRFamide-induced contractions were reduced by 70% and 90%, respectively, in larvae with reduced expression of the Drosophila Fmrf receptor (FR) either ubiquitously or specifically in muscle tissue, compared with the response in control larvae in which expression was not manipulated. No such effect occurred in larvae with reduced expression of this gene only in neurons. The myogenic effects of DPKQDFMRFamide do not appear to be mediated through either of the two Drosophila myosuppressin receptors (DmsR-1 and DmsR-2). DPKQDFMRFamide-induced contractions were not reduced in Ala1 transgenic flies lacking activity of calcium/calmodulin-dependent protein kinase (CamKII), and were not affected by the CaMKII inhibitor KN-93. Peptide-induced contractions in the mutants of the phospholipase C-β (PLCβ) gene (norpA larvae) and in IP3 receptor mutants were similar to contractions elicited in control larvae. The peptide failed to increase cAMP and cGMP levels in Drosophila body wall muscles. Peptide-induced contractions were not potentiated by 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, and were not antagonized by inhibitors of cAMP-dependent or cGMP-dependent protein kinases. Additionally, exogenous application of arachidonic acid failed to induce myogenic contractions. Thus, DPKQDFMRFamide induces contractions via a G-protein coupled FMRFamide receptor in muscle cells but does not appear to act via cAMP, cGMP, IP3, PLC, CaMKII or arachidonic acid.

Endothelin-1 and H2O2-induced signaling in vascular smooth muscle cells : modulation by CaMKII and Nitric oxide

L’endothéline-1 (ET-1) est un peptide vasoactif extrêmement puissant qui possède une forte activité mitogénique dans les cellules du muscle lisse vasculaire (VSMCs). Il a été démontré que l’ET-1 est impliquée dans plusieurs maladies cardio-vasculaires, comme l’athérosclérose, l'hypertension, la resténose après l'angioplastie, l’insuffisance cardiaque et l'arythmie. L’ET-1 exerce ses effets via plusieurs voies de signalisation qui incluent le Ca2+, les protéines kinases activées par les mitogènes (MAPKs) y compris les kinases régulées par les signaux extracellulaires (ERK1/2) et la voie de la phosphatidylinositol 3-kinase (PI-3K)/protein kinase B (PKB). Plusieurs études ont démontré que les dérivés réactifs de l'oxygène (ROS) peuvent jouer un rôle important dans la signalisation d’ERK1/2 et de PKB induite par plusieurs facteurs de croissance et hormones. Nous avons précédemment montré que l'ET-1 produit des ROS qui agissent comme médiateur de la signalisation cellulaire induite par l’ET-1. Le peroxyde d’hydrogène (H2O2), une molécule qui appartient à la famille des ROS, peut activer les voies de la MAPK et de la PKB dans les VSMCs. Par ailleurs, nos résultats suggèrent également que le Ca2+ et la calmoduline (CaM) sont essentiels pour la phosphorylation d’ERK1/2, de p38 et de PKB induite par le H2O2 dans les VSMCs. La Ca2+/CaM-dependent protein kinases II (CaMKII) est une sérine/thréonine protéine kinase multifonctionnelle activée par le Ca2+/CaM. Il a été montré que la CaMKII est impliquée dans les voies de signalisation induite par le H2O2 dans les cellules endothéliales. Cependant, le rôle de la CaMKII dans la phosphorylation d’ERK1/2, de PKB et de la proline-rich tyrosine kinase 2 (Pyk2) induite par l’ET-1 et le H2O2, de même que son rôle dans l’effet hypertrophique et prolifératif de l’ET-1 dans les VSMCs demeure inexploré. Le monoxyde d’azote (NO) est une molécule vasoactive impliquée dans la régulation de plusieurs réponses hormonales. Le NO peut moduler la signalisation contrôlant la croissance cellulaire induite par plusieurs agonistes d’où son rôle protecteur dans le système vasculaire. Des études ont montré que le NO peut inhiber la voie de Ras/Raf/ERK1/2 et la voie de PKB induite par le facteur de croissance endothélial (EGF) et l’angiotensine II (Ang II). Beaucoup d’autres travaux ont mis en évidence un cross-talk entre les voies de signalisation activées par l’ET-1 et le NO. La capacité du NO à inhiber la signalisation intracellulaire induite par l’ET-1 dans les VSMCs demeure inconnue. Le travail présenté dans cette thèse vise à déterminer le rôle du système Ca2+-CaM-CaMKII dans la phosphorylation d’ERK1/2, de PKB et de Pyk2 induite par l’ET-1 et le H2O2 ainsi que son rôle dans la croissance et la prolifération cellulaire induites par l’ET-1 dans les VSMCs. Nous avons également testé le rôle du NO dans la phosphorylation d’ERK1/2, de PKB et de Pyk2 ainsi que la synthèse protéique induite par l’ET-1. Dans la première partie de notre étude, nous avons examiné le rôle de la CaMKII dans la phosphorylation d’ERK1/2 et de PKB induite par l’ET-1 dans les VSMCs en utilisant trois approches différentes i.e. l'usage d'inhibiteurs pharmacologiques, un peptide auto-inhibiteur de la CaMKII (CaMKII AIP) et la technique de siRNA. Nous avons démontré que la CaMKII est impliquée dans la phosphorylation d’ERK1/2 et de PKB induite par l’ET-1 dans les VSMCs. Des études précédentes ont montré à l’aide d’inhibiteurs pharmacologiques comme le KN-93 que l'Ang II et les agents induisant une augmentation de la concentration en Ca2+ intracellulaire comme l’ionomycine, provoquent la phosphorylation d’ERK1/2 via la CaM dans les VSMCs. Cependant, en utilisant différentes approches, nos études ont montré pour la première fois une implication de la CaMKII dans la phosphorylation d’ERK1/2 et de PKB induite par l’ET-1 dans les VSMCs. Nous avons également rapporté pour la première fois, un rôle crucial de la CaMKII dans la pathophysiologie vasculaire associée à l’ET-1 puisque l’activation de la CaMKII joue un rôle important dans l’hypertrophie et la croissance cellulaire. Dans la deuxième partie, à la lumière des études précédentes qui montraient que les ROS agissent comme médiateurs de la signalisation induite par l’ET-1 dans les VSMCs, nous avons examiné si la CaMKII est également impliquée dans l’activation des voies d’ERK1/2 et de PKB induite par le H2O2. En utilisant des approches pharmacologiques et moléculaires, nous avons montré, comme pour l’ET-1, que la CaMKII joue un rôle critique en amont de la phosphorylation d’ERK1/2, de PKB et de Pyk2 induite par le H2O2. Nous avons précédemment montré que la transactivation du récepteur de type I de l’insulin-like growth factor (IGF-1R) est nécessaire à l’activation de PKB induite par le H2O2. Pour cette raison, nous avons examiné l'effet de l'inhibition de la CaMKII par l’inhibiteur pharmacologique ou par le knock-down de la CaMKII sur la phosphorylation d’IGF-1R induite par le H2O2. Les résultats démontrent que la CaMKII joue un rôle critique en amont de la phosphorylation d’ERK1/2, de PKB et d’IGF-1R induite par le H2O2. Dans la troisième partie de notre étude, nous avons également examiné le mécanisme moléculaire par lequel le NO exerce ses effets anti-mitogéniques et anti-hypertrophiques dans la signalisation induite par l’ET-1. En testant l'effet de deux différents donneurs de NO (S-nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside (SNP)) et un inhibiteur de NO synthase, le N (G)-nitro-L-arginine methyl ester (L-NAME) dans la phosphorylation d’ERK1/2, de PKB et de Pyk2 induite par l’ET-1, nous avons observé que le NO a un effet inhibiteur sur la signalisation induite par l’ET-1 dans les VSMCs. Par ailleurs, le 8-Br-GMPc, un analogue du GMPc, a un effet similaire à celui des deux donneurs du NO, tandis que l’oxadiazole quinoxaline (ODQ), un inhibiteur de la guanylate cyclase soluble, inverse l'effet inhibiteur du NO. Nous concluons que le NO diminue la phosphorylation d’ERK1/2, de PKB et de Pyk2 induite par l’ET-1 d’une manière dépendante du GMPc. Le NO inhibe aussi les effets hypertrophiques de l’ET-1 puisque le traitement avec le SNAP diminue la synthèse des protéines induite par l’ET-1. En résumé, les études présentées dans cette thèse démontrent que l’ET-1 et le H2O2 sont des activateurs de la phosphorylation d’ERK1/2, de PKB et de Pyk2 dans les VSMCs et que la CaMKII s’avère nécessaire pour ce processus, en agissant en amont de l’activation de IGF-1R induite par le H2O2 dans les VSMCs. Elles montrent également que le NO inhibe la phosphorylation d’ERK1/2, de PKB et de Pyk2 induite par l’ET-1. Enfin, nos travaux suggèrent aussi que l’activation de la CaMKII stimule la synthèse des protéines et de l’ADN induites par l’ET-1 alors que le NO inhibe la synthèse des protéines induite par ET-1. Mots clés: Endothéline ; Peroxyde d'hydrogène ; CaMKII ; Monoxyde d’azote ; Système vasculaire ; PKB; ERK1/2; IGF-1R; Hypertrophie.

Développement d’une méthode de simulation multi-échelle pour l’étude des grandes transformations dans les protéines

Les films de simulations qui accompagnent le document ont été réalisés avec Pymol.