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Thesis (M App Sc) -- University of South Asutralia, 1991

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Ubiquitylation is a highly controlled post-translational modification of proteins, in which proteins are conjugated either with monoubiquitin or polyubiquitin chains. Ubiquitin modifications on target proteins are recognized by ubiquitin-binding domains, which are found in several effector proteins. In this study, we describe for the first time how ubiquitin controls the function of the Toll-interacting protein (Tollip), which is an effector protein in the innate immune signaling pathway and an adaptor protein for endosomal trafficking. We have demonstrated that the central C2 domain of Tollip preferentially interacts with phosphoinositides with moderate affinity. Remarkably, we found that ubiquitin modulates Tollip's lipid binding. We have observed an ubiquitin dose-dependent inhibition of binding of Tollip to phosphoinositides and it does so specifically by blocking Tollip C2 domain-phosphoinositide interactions. This led us to discover that the Tollip C2 domain is a novel ubiquitin-binding domain. In addition, we have biophysically characterized the association of the Tollip CUE domain to ubiquitin and compared it with Tollip C2 domain-ubiquitin binding. The Tollip CUE domain reversibly binds ubiquitin with affinity higher than C2 domain and at a site that overlaps with that corresponding to the Tollip C2 domain. We have also found that ubiquitin binding to dimeric Tollip CUE domain induces a drastic conformational change in the protein, leading to the formation of a heterodimeric Tollip CUE-ubiquitin complex. These data suggest that ubiquitin binding to the Tollip C2 and CUE domains and ubiquitin-mediated dissociation of CUE dimer reduces the affinity of the Tollip protein for endosomal phosphoinositides, allowing Tollip's cytoplasmic sequestration. Overall, our findings will provide the structural and molecular basis to understand how Tollip works inside the cell and commit itself to cytosolic signalling or endosomal trafficking in a ligand dependent manner. / Ph. D.

Les neutrophiles participent à la défense de l'hôte en phagocytant les agents pathogènes et en les détruisant via notamment la production de formes réactives de l'oxygène (FRO). Les FRO sont produites par un complexe multi- protéique :la NADPH oxydase (NOX2). Celle-ci peut s’assembler à la membrane du phagosome lors de la phagocytose mais aussi à la membrane plasmique lors de la stimulation des neutrophiles par des agents bactériens ou des médiateurs de l’inflammation. La NADPH oxydase est une arme à double tranchant; une activation excessive ou inappropriée de la NADPH oxydase génère un stress oxydant, facteur aggravant des nombreuses pathologies. Cette enzyme doit donc être finement régulée. La NADPH oxydase est activée lorsque les sous-unités cytosoliques de NOX2 (p67phox, p47phox, p40phox) et la petite GTPase Rac s’assemblent avec les sous-unités membranaires (p22phox et gp91phox) à la membrane phagosomale ou plasmique. P67phox régule le flux d'électrons qui transite via gp91phox du NADPH à O2.-. Des travaux récents indiquent que les phospholipides anioniques contribueraient à la régulation de la NADPH oxydase. De plus, Les protéines organisatrices p40phox et p47phox possèdent des domaines de liaison à ces phosphoinositides : p40phox peut se lier au phosphatidylinositol 3-phosphate (PI(3)P) et p47phox au phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). Nous avons donc voulu comprendre le rôle des ces phospholipides dans la régulation de la NADPH oxydase. Dans un premier temps nous nous sommes intéressés au rôle du PI(3)P, présent au phagosome après la fermeture de celui-ci, dans l’activation de la NADPH oxydase. Nos données indiquent que p40phox fonctionne comme un adaptateur, PI(3)P dépendant, permettant de maintenir p67phox dans le complexe de la NADPH oxydase. Le PI(3)P agit comme un « timer » pour l'activation de la NADPH oxydase au phagosome. Nous avons ensuite voulu examiner le rôle du PI(3,4)P2 dans la régulation de la NADPH oxydase à la membrane plasmique. Ce lipide est formé à la membrane plasmique par phosphorylation du PI(4)P par la PI3K de classe I lors de l’activation des neutrophiles. Nous avons montré que, l'activité PI3K de classe I est nécessaire pour maintenir l’activation, intégrine-dépendante, de la NADPH oxydase à la membrane plasmique. / The NADPH oxidase of the professional phagocyte is essential for the immune system. The phagocyte NADPH oxidase, NOX2, catalyze the reduction of molecular oxygen to superoxide. Superoxide is transformed rapidly into other reactive oxygen species (ROS) which play a critical role in the killing of pathogens in host defense. Indeed neutrophils, the first cells that arrive at the site of infections, engulf pathogens in a process called phagocytosis. The production of reactive oxygen species is then triggered by the NADPH oxidase in the phagosome. The importance of ROS production is demonstrated by the recurrent bacterial and fungal infections that face patients who lack functional NADPH oxidase as in the rare genetic disorder known as the chronic granulomatous disease (CGD). Upon stimulation by bacterial peptide or in some pathological conditions, NADPH oxidase can also be activated at the phagocyte plasma membrane producing ROS in the extracellular medium. So, an excessive or inappropriate NADPH oxidase activation generates oxidative stress involve in chronic inflammation, cardiovascular disease and neurodegenerative disease. The NADPH oxidase activity should be tightly regulated. The activity of the enzyme is the result of the assembly of cytosolic subunits (p47phox, p67phox, p40phox and Rac2) with membranous subunits (gp91phox and p22phox). P67phox regulates the electron flow through gp91phox from NADPH to oxygen leading to the formation of superoxide. Recent data indicate that the anionic phospholipids are important for the NADPH oxidase regulation. Moreover, p40phox and p47phox bear a PX domain that binds respectively phosphatidylinositol3-phosphate (PI3P) and phosphatidylinositol (3,4)-bisphosphate(PI(3,4)P2). Our objective was to decipher the importance of these phosphoinositides on the NADPH oxidase activity. We first examined the role of PI3P, which is present on the cytosolic leaflet of phagosome after its sealing, in NADPH oxidase activation. Our data indicate that p40phox works as a late adaptor controlled by PI3P to maintain p67phox in the NADPH oxidase complex. Thus, PI3P acts as a timer for NADPH oxidase assembly. We then examined the role of PI(3,4)P2 in the activation of the NADPH oxidase assembled at the plasma membrane. PI(3,4)P2 and PI(3,4,5)P3 are formed at the plasma membrane, upon neutrophil activation, by phosphorylation by Class I PI3K of respectively PI4P and PI(4,5)P2. We found that class I PI3K activity is required to maintain the integrin-dependent activation of NADPH oxidase at the plasma membrane.

Phosphatidylinositol mannosides (PIMs) isolated from mycobacteria have been identified as an important class of glycolipids that possess significant immune modulating properties. To provide discrete synthetic compounds for biological assay, this thesis describes the syntheses of three PIM molecules, namely dipalmitoyl PIM2 (12), PIM4 (84), and PIM6 (108), and two PIM2 analogues designed for increased stability, PIM2ME (147) and PIM2MA (148). The synthesis of all of these molecules involved mannosylation of 1-O-allyl-3,4,5-tri-O-benzyl-D-myo-inositol (22), which was prepared from methyl α-D-glucopyranoside in 8% yield over 8 steps, using a Ferrier reaction strategy. A common intermediate, 3,4,5-tri-O-benzyl-2,6-di-O-(2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl)-D-myo-inositol (9), was used for the syntheses of 12, 147, and 148. This compound was prepared by bis-mannosylation of the C-1 and C-6 hydroxyl groups of 22 with 2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl trichloroacetimidate (63) to give, after protecting group manipulations, the α,α-pseudo-trisaccharide 9 in 37% over 4 steps. The selectivity of the desired α,α-product was found to be increased by the selection of Et₂O as the solvent for the glycosylation reaction. The C-1 hydroxyl group of 9 was coupled to benzyl (1,2-di-O-palmitoyl-sn-glycero)-diisopropylphosphoramidite (28) using 1H-tetrazole. Global debenzylation of the resulting product gave PIM2 (12) in 23% yield over 6 steps from 22. In a similar fashion 9 was coupled to 1-O-hexadeconyl-2-O-hexadecyl-sn-glycero-3-O-benzyl-(N,N-diisopropyl)-phosphoramidite (156), and subsequent deprotection gave PIM2ME (147) in 30% yield over 2 steps from 9. Coupling of 9 with 2-deoxy-1-O-hexadeconyl-2-O-hexadeconylamino-sn-glycero-3-O-benzyl-(N,N-diisopropyl)-phosphoramidite (172) and subsequent deprotection gave PIM2MA (148) in 47% yield over 2 steps from 9. A modified approach was required for the syntheses of PIM4 (84) and PIM6 (108). A selective glycosylation of the C-6 hydroxyl of 22 with an orthogonally protected mannose donor would allow extension of the manno-oligosaccharide in a 2+3 or 4+3 glycosylation strategy required to build the pseudo-pentasaccharide or pseudo-heptasaccharide core of 84 or 108 respectively. Sequential mannosylation of 22, firstly at the more reactive C-6 hydroxyl, with 2-O-acetyl-3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-α-D-mannopyranosyl trichloroacetimidate (85), was followed by mannosylation at the C-2 hydroxyl with 63. Removal of the silyl protecting group followed by a 2+3 coupling with the dimannoside donor, 2-O-acetyl-6-O-(2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-3,4-di-O-benzyl-α-D-mannopyranosyl trichloroacetimidate (95), gave a pseudo-pentasaccharide intermediate. Protecting group manipulations followed by coupling of the of the C-1 hydroxyl group of the inositol ring to phosphoramidite 28, and a global debenzylation, gave PIM4 (84) in 6% yield over 9 steps from 22. During the synthesis of PIM6 (108), thioglycosylation chemistry was explored and found to be comparable to reactions with trichloroacetimidate donors. Similar methodology was used for the synthesis of PIM6 (108) as had previously been carried out for the synthesis of PIM4 (84). Mannosylation at the more reactive C-6 hydroxyl of 22 with either phenyl 2-O-benzoyl-3,4-di-O-benzyl-6-O-triisopropylsilyl-1-thio-α-D-mannopyranoside (112) or 2-O-benzoyl-3,4-di-O-benzyl-6-O-triisopropylsilyl-α-D-mannopyranosyl trichloroacetimidate (113), was followed by mannosylation at the C-2 hydroxyl with 63. Removal of the silyl group followed by a 4+3 coupling with either of the tetramannoside donors, phenyl (2-O-benzoyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1[to]2)-(3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1[to]2)-(3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1[to]6)-2-O-benzoyl-3,4-di-O-benzyl-1-thio-α-D-mannopyranoside (109) or (2-O-benzoyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1[to]2)- (3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1[to]2)-(3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1[to]6)-2-O-benzoyl-3,4-di-O-benzyl-α-D-marmopyranosyl trichloroacetimidate (131) gave a gave a pseudo-heptasaccharide intermediate. Protecting group manipulations followed by coupling of the of the C-1 hydroxyl group of the inositol ring to phosphoramidite 28, and a global debenzylation, gave PIM6 (108) in 9% yield over 9 steps from 22. To aid characterisation of 108, a sample was deacylated to afford dPIM6 (144) which gave the same spectral data as a sample from a natural source. The compounds PIM2 (12), PIM4 (84), PIM2ME (147), and PIM2MA (148) were assayed for adjuvant activity and were found to have comparable activity to fractions isolated from natural sources. The analogue PIM2ME (147) gave the best results and is currently undergoing further development.

Long-term stimuli of many systems leads to decreased cellular responsiveness, or desensitization. We characterized the desensitization of angiotensin II (Ang 11)-mediated inositol phospholipid (IP) hydrolysis in cultured human aortic smooth muscle cells (HASMC). Although it has been suggested that the desensitization induced by long-term Mg II exposure may result partially from down-regulation of Ang II receptor, this is not sufficient to explain fully desensitization in many systems. Post-receptor desensitization of IP hydrolysis may also result from phosphorylation or changes in protein levels of the effector enzyme, PLC-β. We identified the major PLC-β isoenzymes expressed by HASMC as PLC-β1 and PLC-β3. Ang II pretreatment reduced IP accumulation induced by Ang II (1μM) in a time-dependent manner. Phorbol ester-12-myristrate-13-acetate (PMA), a protein kinase C (PKC) activator, also reduced Ang II-stimulated IP accumulation. These results suggest that PKC activation may negatively regulate Ang II-stimulated IP signaling in HASMC, similar to rat cells. In addition, PKC also reduced IP accumulation stimulated by A1F₄⁻, directly activating the G protein. It suggests that the majority of PKC-induced desensitization of Ang II-stimulated IP signaling occurs downstream of the Ang II receptor in HASMC. However, both PLC-β1 and PLC-β3, expected candidates for PKC phosphorylation, were phosphorylated independently of PKC activation or inhibition, indicating that PKC might not be involved in direct phosphorylation of PLC-β1 and PLC-β3. Furthermore, PLC-β1, but not PLC-β3, was highly phosphorylated under basal conditions, suggesting that PLC-β1 and PLC-β3 may play different roles in IP signaling in HASMC. / Graduation date: 2003

Les plaquettes jouent un rôle majeur dans l'hémostase mais également dans les maladies cardiovasculaires qui représentent une des principales causes de mortalité dans les pays industrialisés. Au cours de l'activation plaquettaire, les phosphoinositides 3-kinases (PI3Ks) de classe I génèrent des seconds messagers lipidiques (D3-phosphoinositides) participant activement à la transmission des signaux en aval des principaux récepteurs plaquettaires. Comme l'inhibition de la PI3Kbeta protège des thromboses occlusives sans augmenter le risque de saignement, cette kinase a été proposée comme nouvelle cible antithrombotique. Toutefois, le rôle de la PI3Kbeta dans des modèles de thrombose in vivo et ex vivo, dans des conditions de forces de cisaillement élevées, restait mal documenté au début de ma thèse. Le rôle de la PI3Kalpha?était également mal connu bien que l'utilisation d'inhibiteurs peu spécifiques suggérait son implication en aval du récepteur GPVI. L'objectif de ma thèse a été d'étudier le rôle de la PI3Kbeta dans la formation du thrombus in vivo et ex vivo, et de caractériser la fonction de la PI3Kalpha plaquettaire. Grâce à des souris présentant une invalidation de la PI3Kbeta ou alpha spécifiquement dans la lignée mégacaryocytaire, j'ai montré que la PI3Kbeta plaquettaire jouait un rôle essentiel dans la stabilité du thrombus dans des conditions de forces de cisaillement élevées. Son inhibition affecte l'activation d'Akt et empêche l'inhibition de son effecteur GSK3 au sein même du thrombus. De façon intéressante, l'inhibition de GSK3 restaure la stabilité du thrombus. Ainsi, la PI3Kbeta joue un rôle critique dans le maintien de l'intégrité du thrombus exposé à des forces de cisaillement élevées et cet effet ne peut être compensé par la PI3Kalpha. D'autre part, l'absence de la PI3Kalpha plaquettaire se traduit par un défaut d'agrégation et d'activation d'Akt lors d'une stimulation de faible intensité de GPVI. In vivo, le thrombus formé par les plaquettes invalidées pour la PI3Kalpha consécutivement à une lésion superficielle des artères, est de plus petite taille que chez les souris sauvages. En condition de flux ex vivo, il est noté un retard de formation du thrombus sur les fibres de collagène suggérant l'implication de la PI3Kalpha dans la phase d'adhésion des plaquettes à la matrice. En effet, des études d'adhésion en flux sur une matrice de facteur de von Willebrand (vWF) montrent que la PI3Kalpha est essentielle pour l'adhésion stable des plaquettes à cette matrice via la signalisation outside-in de l'intégrine alphaIIbbeta3. En conclusion, mes travaux de thèse mettent à jour un rôle spécifique des PI3Ks alpha et beta de classe I dans les plaquettes. La PI3Kbeta? est cruciale dans la régulation de la croissance et de la stabilité du thrombus dans des conditions de forces de cisaillement élevées, tandis que la PI3Kalpha?est requise dans la phase initiale d'adhésion des plaquettes sur une matrice de vWF. Ce travail apporte des nouvelles données qui seront utiles notamment dans le contexte du développement d'inhibiteurs sélectifs de PI3Ks en thérapie anticancéreuse et antithrombotique. / Platelets play a major role in cardiovascular diseases which is one principal cause of worldwide death. Class I phosphoinositide 3-kinases (PI3Ks) are important signaling enzymes in the process of blood platelet activation, producing lipid second messengers (D3-phosphoinositides) that are actively involved downstream of major platelet receptors. PI3Kbeta has been proposed as a potential drug target to treat arterial thrombosis. Indeed, inhibition of this lipid kinase leads to protection against occlusive thrombosis without bleeding risk. Nevertheless, nothing is known regarding the role of this kinase in vivo and ex vivo during growth, stabilization, and resistance upon elevation of shear rate. One study, using pharmacological inhibitors, has suggested that both PI3Kalpha and beta are required, in a non redundant way, for full platelet activation through collagen receptor GPVI, but the role of PI3Kalpha still remains elusive in platelets. The aim of my thesis was to study the role of PI3Kbeta during thrombus formation in vivo and ex vivo, and to characterize the role of PI3Kalpha in platelets. For that, I used pharmacological approach and mice with selective deletion of PI3Kbeta?or?PI3Kalpha in the megakaryocyte lineage (PF4-Cre/p110betaflox/flox and PF4-Cre/p110alphaflox/flox). I showed that PI3Kbeta is essential for thrombus growth and stability at high shear rates. Within the growing platelet thrombus, PI3Kbeta inactivation impairs the activating phosphorylations of Akt and the inhibitory phosphorylation of GSK3. In line with these data, pharmacological inhibition of GSK3 restores thrombus stability. Thus, platelet PI3Kbeta has a critical role in maintaining the integrity of the formed thrombus upon elevation of shear rate. In this condition, I showed that PI3Kbeta absence cannot be compensated by PI3Kalpha. In vitro, PI3Kalpha depletion in platelets leads to a light defect of aggregation and Akt activation in response to CRP showing implication of PI3Kalpha downstream GPVI. I observed that, in vivo, thrombi formed by PI3Kalpha depleted platelets after superficial lesion of mesenteric arterial are smaller, and ex vivo, thrombus formation under flow conditions on collagen matrix is delayed. Furthermore, perfusion of these platelets on von Willebrand factor matrix (vWF) shows that PI3Kalpha is required for stable adhesion of platelets through "outside in" signaling. Altogether, these results show an involvement of PI3Kalpha in the course of early step of platelet adhesion. In conclusion, my thesis work highlights an isoform specific role of PI3Ks in platelets. PI3Kbeta is crucial in the regulation of thrombus growth and stability at a high shear rate and PI3Kalpha is required in initial stages of platelet adhesion. Since class I PI3Ks selective inhibitors are under development as cancer treatment, these results may help to anticipate the potential side effects of such treatment on haemostasis.

No description available.