No description available.

Toll-like receptors (TLRs) are the main components of the innate immunity. Pathogen-activated TLRs trigger a cytoplasmic signaling cascade through adaptor proteins, with the first being the TIR domain-containing adaptor protein (TIRAP). TIRAP contains a TIR domain, which associates with TLRs and other adaptor proteins; and a N-terminal phosphoinositide-binding motif (PBM) that mediates the membrane recruitment of TIRAP. Upon ligand activation, TLRs are recruited to the phosphoinositide (PIP)-enriched region in the membrane, where TIRAP recruits other adaptors to the membrane to activate TLR signaling pathway. To investigate the mechanism of membrane targeting of TIRAP and the basis for its regulation, I functionally and structurally characterized TIRAP and its PBM using biophysical approaches. I show that TIRAP PBM adopts helical structural in dodecylphosphocholine (DPC) micelles and other membrane mimics. NMR studies reveal that TIRAP PBM binds PIPs following a fast exchange regime with a moderate affinity through two conserved basic termini. Mutation of these two basic regions abolishes PIPs binding without distorting the helical structure of the peptide. Solution NMR structure of TIRAP PBM exhibits a central relatively hydrophobic helix surrounded by the flexible N- and C-termini. Paramagnetic studies indicate that the helix is close to the micelle core, whereas two termini are located on the micellar surface. Nuclear spin relaxation experiments indicate that the two termini of TIRAP PBM become more ordered when bound to PIP, thus, we propose that the central helix in PBM is responsible for membrane insertion, whereas the two sets of basic residues interact with PIPs to stabilize TIRAP's membrane interaction. Phosphomimetic mutation of Thr28 to Asp (T28D) as well as phosphorylation in Thr28 inhibit TIRAP PBM's binding to phosphoinositides by distorting the central helical structure of the peptide. More importantly, TIRAP T28D disrupt its subcellular localization in vivo. Thus, phosphorylation can impair proper insertion of TIRAP at the plasma membrane through PBM and, consequently, it may represent the first signal that promotes TIRAP degradation. / Ph. D.

Le carcinome hépatocellulaire (CHC) et le cholangiocarcinome (CCA) sont 2 types de cancer primitif du foie. Le CHC est le plus fréquent, cependant l’incidence du CCA augmente partout dans le monde avec un diagnostic difficile, un mauvais pronostic et des thérapies très limitées. Ce travail avait pour objectif d'identifier des cibles pour le diagnostic et la thérapeutique du CCA. Il est basé sur l'étude de la septine 9 et des phosphoinositides (PIs). La septine 9 appartient à une famille de GTPases qui participent à l’organisation des microtubules et du cytosquelette d’actine. Les septines sont impliquées dans la cytokinèse, le trafic vésiculaire et la polarité cellulaire, elles sont aussi des partenaires importantes des PIs. Pour déterminer le rôle de la septine 9 dans le CCA nous nous sommes intéressés à son interaction avec les PIs et avec l’inhibiteur de l’inducteur et activateur de la transcription 1 (PIAS1) qui a été décrite comme une protéine pouvant agir comme une SUMO ligase pour les septines. Nous avons étudié l’expression de la septine 9 et de PIAS1 dans le CCA et le CHC. Nous avons mis en évidence un mécanisme original par lequel, la production du PtdIns5P (Phosphatidylinositol -5-phosphate) permet un recrutement de la septine 9, la stabilisation des microtubules et le transport de PIAS1 du cytoplasme vers le noyau. Il démontre un rôle important des septines en association avec les PIs dans le trafic. De plus, nous avons montré que la septine 9 est un régulateur de la signalisation de l’interféron γ qui agit au niveau de la phosphorylation de STAT1 et l’entrée de PIAS1 dans le noyau. Ce travail peut constituer une nouvelle piste pour la recherche des thérapies ciblées en immunothérapie dans le traitement de ce cancer. / Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are two types of primary liver cancer. HCC is the most frequent, however the incidence of CCA increases throughout the world with a difficult diagnosis, poor prognosis and very limited therapies. The objective of this work was to identify targets for the diagnosis and treatment of CCA. It is based on the study of septin 9 and phosphoinositides (PIs). Septin 9 belongs to a family of GTPases that participate in the organization of microtubules and the actin cytoskeleton. Septins are involved in cytokinesis, vesicular trafficking and cellular polarity and are also important partners of PIs. To determine the role of septin 9 in the CCA, we investigated its interaction with PIs and with Protein inhibitory of activated STAT1 (PIAS1), which has been described as a SUMO ligase for septins. We studied the expression of septin 9 and PIAS1 in CCA and CHC. We have demonstrated an original mechanism by which la production of PtdIns5P allows the recruitment of septin 9, the stabilization of microtubules and the transport of PIAS1 from the cytoplasm to the nucleus. It demonstrates an important role of the septins in association with the PIs in trafficking. Besides, we have shown that septin 9 is a regulator of interferon γ signaling which acts at the level of the phosphorylation of STAT1 and the entry of PIAS1 into the nucleus. This work can constitute a new avenue for the research of targeted immunotherapy for this cancer.

No description available.

Phosphoinositide 3-kinases (PI3Ks) are a family of enzymes that catalyse the synthesis of different lipid second messengers, regulating a plethora of intracellular functions. Deregulation of their signalling pathway has different functional consequences, and it has been associated with a variety of human diseases. The existence of eight distinct isoforms, divided into three classes, has raised in the past many questions on whether and to which extent their role was redundant or overlapping. The study of their intracellular signalling pathways and cellular functions is crucial, because some of these isoforms have been identified as important therapeutic targets. The most investigated PI3Ks belong to the class I subfamily, and they have a well established role in the regulation of cell growth, survival and proliferation. In the past years, attention and research efforts focussed on class I isoforms and alteration of their signalling pathways, one of the most common causes of cancer. More recently, evidence indicated that the least investigated class II PI3Ks have different intracellular roles. This work focussed on the class II isoform PI3K-C2α, the study of its intracellular function(s) in pancreatic β cells and the implications of its inhibition through downregulation in pancreatic β cells homeostasis. It was reported that PI3K-C2α has a crucial role insulin granules exocytosis. This study has demonstrated that this enzyme synthesises the lipid product PtdIns3P specifically at the plasma membrane of pancreatic β cells upon depolarisation of the plasma membrane and stimulation of insulin secretion. Moreover, the data herein presented indicated for the first time that glucose-induced activation of PI3K-C2α is able to protect β cells from cell death induced by nutrients deprivation. Analysis of the intracellular pathways stimulated by glucose indicated that PI3K-C2α is able to modulate the activity of mTOR and its downstream effectors, key regulators of cell proliferation and growth. Importantly, the activation of mTOR pathway upon glucose does not seem to involve the activation of the upstream regulator Akt or class I PI3K, suggesting a novel intracellular pathway stimulated by glucose.

The eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a protein normally expressed only in the brain, heart and skeletal muscle. eEF1A2 is likely to be a breast and ovarian cancer oncogene based on its high expression in these malignancies and its in vitro transforming capacity . The goal of my thesis is to understand eEF1A2’s role in oncogenesis. In order to determine if eEF1A2 was a prognostic marker for ovarian cancer, we examined eEF1A2 expression in 500 primary human ovarian tumours. We show that eEF1A2 is highly expressed in approximately 30% of ovarian tumours. In serous cancer, high expression of eEF1A2 was associated with an increased 20-year survival probability. Expression of eEF1A2, in a clear cell carcinoma cell line, SK-OV-3, increased the cells ability to form spheroids in hanging drop culture, enhanced in vitro proliferative capacity, increased stress fiber formations, and reduced cell-cell junction spacing. Expression of eEF1A2 did not alter sensitivity to anoikis, cisplatin, or taxol. In order to examine the role of eEF1A2 in breast cancer, we used a three-dimensional culture system. The ability to disrupt the in vitro morphogenesis of breast cells cultured on reconstituted basement membranes is a common property of breast oncogenes. I found that phosphatidylinositol 4-kinase (PI4KIIIβ), a lipid kinase that phosphorylates phosphatidylinositol (PI) to PI(4)P, disrupts in vitro mammary acinar formation. The PI4KIIIβ protein localizes to the basal surface of acini created by the human MCF10A cells and ectopic expression of PI4KIIIβ induces multi-acinar formation. Expression of the PI4KIIIβ activator, eEF1A2, also causes a multi-acinar phenotype. Ectopic expression of PI4KIIIβ or eEF1A2 alters PI(4)P and PI(4,5)P2 localization, indicating a role for these lipids in acinar development. Therefore, eEF1A2 is highly expressed in ovarian carcinomas and its expression enhances cell growth in vitro. eEF1A2 expression is likely to be a useful ovarian cancer prognostic factor in ovarian patients with serous tumours. Furthermore, PI4KIIIβ and eEF1A2 both have an important role in the disruption of three-dimensional morphogenesis of MCF10A cells. Additionally, PI4KIIIβ and eEF1A2 likely have an important role in mammary neoplasia and development and could be anti-cancer targets.

The eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a protein normally expressed only in the brain, heart and skeletal muscle. eEF1A2 is likely to be a breast and ovarian cancer oncogene based on its high expression in these malignancies and its in vitro transforming capacity . The goal of my thesis is to understand eEF1A2’s role in oncogenesis. In order to determine if eEF1A2 was a prognostic marker for ovarian cancer, we examined eEF1A2 expression in 500 primary human ovarian tumours. We show that eEF1A2 is highly expressed in approximately 30% of ovarian tumours. In serous cancer, high expression of eEF1A2 was associated with an increased 20-year survival probability. Expression of eEF1A2, in a clear cell carcinoma cell line, SK-OV-3, increased the cells ability to form spheroids in hanging drop culture, enhanced in vitro proliferative capacity, increased stress fiber formations, and reduced cell-cell junction spacing. Expression of eEF1A2 did not alter sensitivity to anoikis, cisplatin, or taxol. In order to examine the role of eEF1A2 in breast cancer, we used a three-dimensional culture system. The ability to disrupt the in vitro morphogenesis of breast cells cultured on reconstituted basement membranes is a common property of breast oncogenes. I found that phosphatidylinositol 4-kinase (PI4KIIIβ), a lipid kinase that phosphorylates phosphatidylinositol (PI) to PI(4)P, disrupts in vitro mammary acinar formation. The PI4KIIIβ protein localizes to the basal surface of acini created by the human MCF10A cells and ectopic expression of PI4KIIIβ induces multi-acinar formation. Expression of the PI4KIIIβ activator, eEF1A2, also causes a multi-acinar phenotype. Ectopic expression of PI4KIIIβ or eEF1A2 alters PI(4)P and PI(4,5)P2 localization, indicating a role for these lipids in acinar development. Therefore, eEF1A2 is highly expressed in ovarian carcinomas and its expression enhances cell growth in vitro. eEF1A2 expression is likely to be a useful ovarian cancer prognostic factor in ovarian patients with serous tumours. Furthermore, PI4KIIIβ and eEF1A2 both have an important role in the disruption of three-dimensional morphogenesis of MCF10A cells. Additionally, PI4KIIIβ and eEF1A2 likely have an important role in mammary neoplasia and development and could be anti-cancer targets.

The eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a protein normally expressed only in the brain, heart and skeletal muscle. eEF1A2 is likely to be a breast and ovarian cancer oncogene based on its high expression in these malignancies and its in vitro transforming capacity . The goal of my thesis is to understand eEF1A2’s role in oncogenesis. In order to determine if eEF1A2 was a prognostic marker for ovarian cancer, we examined eEF1A2 expression in 500 primary human ovarian tumours. We show that eEF1A2 is highly expressed in approximately 30% of ovarian tumours. In serous cancer, high expression of eEF1A2 was associated with an increased 20-year survival probability. Expression of eEF1A2, in a clear cell carcinoma cell line, SK-OV-3, increased the cells ability to form spheroids in hanging drop culture, enhanced in vitro proliferative capacity, increased stress fiber formations, and reduced cell-cell junction spacing. Expression of eEF1A2 did not alter sensitivity to anoikis, cisplatin, or taxol. In order to examine the role of eEF1A2 in breast cancer, we used a three-dimensional culture system. The ability to disrupt the in vitro morphogenesis of breast cells cultured on reconstituted basement membranes is a common property of breast oncogenes. I found that phosphatidylinositol 4-kinase (PI4KIIIβ), a lipid kinase that phosphorylates phosphatidylinositol (PI) to PI(4)P, disrupts in vitro mammary acinar formation. The PI4KIIIβ protein localizes to the basal surface of acini created by the human MCF10A cells and ectopic expression of PI4KIIIβ induces multi-acinar formation. Expression of the PI4KIIIβ activator, eEF1A2, also causes a multi-acinar phenotype. Ectopic expression of PI4KIIIβ or eEF1A2 alters PI(4)P and PI(4,5)P2 localization, indicating a role for these lipids in acinar development. Therefore, eEF1A2 is highly expressed in ovarian carcinomas and its expression enhances cell growth in vitro. eEF1A2 expression is likely to be a useful ovarian cancer prognostic factor in ovarian patients with serous tumours. Furthermore, PI4KIIIβ and eEF1A2 both have an important role in the disruption of three-dimensional morphogenesis of MCF10A cells. Additionally, PI4KIIIβ and eEF1A2 likely have an important role in mammary neoplasia and development and could be anti-cancer targets.

Universiẗat, Diss., 2000--Bonn.

Le PtdIns5P est un phosphoinositide très peu connu et émerge dernièrement en tant qu’important nouveau second messager. D’ailleurs, l’augmentation artificielle de PtdIns5Pdans les cellules épithéliales, favorise la voie pro-oncogénique Phosphoinositide 3-kinase/AKT. Plus récemment, il a été fait état d’un possible rôle du PtdIns5P dans le processus de lymphomagenèse.Notre laboratoire étudie depuis de nombreuses années les voies de signalisations importantes de l’activation lymphocytaire T et notamment la voie pro-oncogénique PI3K. Le laboratoire s’est tourné notamment vers l’étude des protéines adaptatrices de la famille Dok(Downstream of Kinase) lors de l’activation lymphocytaire T.Ainsi le but de ce travail a été dans un premier temps l’étude des protéines adaptatrices Dok-1 et Dok-2 durant l’activation lymphocytaire T. Plus précisément, nous nous sommes focalisés sur le rôle de leur domaine pleckstrin homology (PH) dans leur fonction inhibitrice durant l’activation lymphocytaire T. De manière intéressante nous avons pu caractériser lePtdIns5P comme un ligand convaincant des domaines PH de Dok-1 et Dok-2 et engendrant leur phosphorylation et donc leur activation. Des dosages de PtdIns5P effectués après une stimulation du récepteur des cellules T (TCR) nous ont permis d’observer une augmentation de PtdIns5P suite à cette activation lymphocytaire T.Dans un second temps nous nous sommes intéressés à caractériser les autres ligands des domaines PH de la famille Dok. De manière intéressante nous avons pu identifier le domaine PH de Dok-5 comme un interacteur fort avec le PtdIns5P. La caractérisation plus précise de l’impact du PtdIns5P, nous a permis de montrer la modulation positive de protéines clés des voies de signalisation intracellulaire telles que les Src kinases et AKT qui sont retrouvées phosphorylées suite à une augmentation de PtdIns5P.L’ensemble de ces résultats faisant du PtdIns5P un nouveau messager de l’activation lymphocytaire T, pouvant contribuer au maintien de l’homéostasie des lymphocytes T / PtdIns5P is a rare phosphoinositide and is starting to emerge as a potential second messenger.Bacterial infection by Shigella flexnerii via the virulence factor IpgD, generates PtdIns5P in thehost cells and induces Akt activation. Recent evidences report that enhanced tyrosinephosphorylation increase cellular PtdIns5P levels and that PtdIns5P could have a role inoncogenesis. Altogether, these data argue for an important role of PtdIns5P in cell signalling.Recently, we demonstrated a PtdIns5P production in T cells upon TCR triggering.Interestingly we showed that Dok-1 and Dok-2 proteins (for Downstream of tyrosine kinase)tyrosine phosphorylation correlated with PtdIns5P increase. These structurally related adaptermolecules contain a pleckstrin homology (PH) domain generally acting as a lipid/proteininteractingmodule. We found that Dok-1/Dok-2 PH domains bind in vitro to PtdIns5P. Thepresence of this PH domain is necessary for the tyrosine phosphorylation of Dok-1 and Dok-2proteins and their negative functions in T cells. Together, our data identify a novel lipid mediatorin T cell signalling and suggest that PtdIns5P could be a new yet uncharacterized secondmessenger in T cell activation.We next focused on a new PtdIns5P binding module, the PH domain Dok-5. Our in vitrodata revealed a stronger interaction between Dok-5 PH and PtdIns5P. We used Dok-5 PHdomain and IpgD as tools to characterize PtdIns5P functional effects on T cells. Our results showthat IpgD expression induces Src-kinase and Akt activation, but not ERK activation andenhances IL-2 promoter activity in T cells. Expression of this PtdIns5P interacting domainblocks IpgD-induced T cell activation and selective signaling molecules downstream of TCRtriggering. Altogether, these data suggest that PtdIns5P may play a sensor function in setting thethreshold of T cell activation and contributing to maintain T cell homeostasis