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

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

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

To understand whether integrin β4 is involved in vascular endothelial cell (VEC) senescence, we examined integrin β4 level changes, as well as P53 and reactive oxygen species (ROS) levels and alterations of phosphatidylcholine-specific phospholipase C (PC-PLC) activity before and after knocking-down integrin β4 by small interfering RNA. We found integrin β4, P53 and ROS levels increased significantly, while Ca2+-independent PC-PLC activity obviously decreased during VEC senescence. On the other hand, integrin β4 down-regulation attenuated the senescence phenotype and reversed Ca2+-independent PC-PLC activity, and P53 and ROS levels. The data suggested that integrin β4 might mediate VEC senescence through depressing Ca2+-independent PC-PLC and elevating the levels of P53 and ROS.

Intravenous administration of ovokinin(2-7), a cleavage peptide derived from ovalbumin, dose-dependently (0.1-5 mg/kg) lowered the mean arterial pressure (MAP) that was not accompanied by a significant change in the heart rate (HR) of urethane-anesthetized rats. The hypotensive effects of ovokinin(2-7) were five orders of magnitude lower compared to that of bradykinin and were largely prevented by pretreatment with the bradykinin B2 receptor antagonist HOE140 (81.6±18.4%) and moderately affected by the B1 receptor antagonist [des-Arg10]-HOE140 (26.3±15.5%). Intracellular Ca2+ levels, as measured by Fur 2-AM, were significantly elevated in cultured aorta smooth muscle cells by ovokinin(2-7). The increases were abolished by HOE140 and unaffected by [des-Arg10]-HOE140. The elevation of intracellular Ca2+ by ovokinin(2-7) was dependent on Ca2+ entry from extracellular space as it was reduced in a Ca2+-free solution. Pretreatment of the cells with the phospholipase C inhibitor U73122 (2 μM) eliminated the Ca 2+ increase by the peptide. PA phosphohydrolase and phospholipase A2 inhibitors significantly reduced the responses as well. Our results show that ovokinin(2-7) modulates cardiovascular activity by interacting with B2 bradykinin receptors.

The objectives of this research were to better characterize the protein signaling complexes that form in response to spermatozoa binding to the bovine oocyte vitelline membrane and to elucidate their potential involvement in oocyte activation. Integrins located on the vitelline membrane of bovine oocytes have been implicated in mediating the sperm-oocyte interaction. Anti-integrin function blocking antibodies and immunofluorescence were utilized in order to reveal that the αV and β1 integrin subunits are essential for fertilization in the bovine and could form the integrin heterodimer involved in the sperm-oocyte interaction. Focal adhesion kinase is localized to focal adhesions and is a key component of signal transduction pathways mediated by integrins. The presence of focal adhesion kinase in bovine oocytes was verified by real-time polymerase chain reaction and immunoprecipitation and the localization of focal adhesion kinase at the site of sperm binding to the oocyte plasma membrane was verified using immunohistochemistry. The inhibition of focal adhesion kinase resulted in fewer cleaved embryos in addition to a reduction in the number of oocytes responding with calcium transients. Phospholipase C isoforms regulate the release of calcium from the endoplasmic reticulum and are known to interact with integrins and focal adhesion kinase. The experiments reported in this dissertation explored the involvement of phospholipase C isoforms endogenous to the oocyte in mediating the calcium release associated with fertilization. Reduction in phospholipase C messenger ribonucleic acid levels for the phospholipase C isoforms γ1 and γ2 resulted in significantly lower cleavage rates compared to the controls. Interestingly, the reduction in messenger ribonucleic acid levels for phospholipase ζ failed to impact cleavage. Maximizing protein levels for the phospholipase C isoforms ζ and γ2 resulted in a significantly higher number of oocytes reaching the 2-cell stage compared to all other treatment groups and not significantly different than the activation control. Together these data illustrate the involvement of the αV and β1 integrin subunits, focal adhesion kinase, and the potential involvement of multiple endogenous phospholipase C isoforms (γ1 and γ2) in bovine oocyte activation. A more complete understanding of the molecular players involved in fertilization could have beneficial impacts for human fertility, assisted reproduction, and improved efficiency of animal somatic cell nuclear transfer.

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Yes / Single nucleotide polymorphisms (SNPs) in PLCE1 and MICB genes increase risk for the development of dengue shock syndrome (DSS). We used Bioinformatics tools to predict alterations at the transcriptional and posttranslational levels driven by PLCE1 and MICB SNPs associated with DSS. Functional and phenotypic analysis conducted to determine deleterious SNPs and impact of amino acid substitution on the structure and function of proteins identified rs2274223 (H1619R) as deleterious to protein coding as it induces structural change in the C2 domain of PLCε, with the mutant residue more positively charged than the wild-type residue (RMSD score, 1.75 Å).Moreover, rs2274223 condenses the chromatinrepressing PLCε expression in DSS. Briefly, this study presents the impact of a single nucleotide transition at SNPs associated with DSS on differential protein binding patterns with PLCE1 and MICB genes and on protein structure modification and their possible role in the pathogenesis of DSS.

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.