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Deoxycytidylate (dCMP) hydroxymethylase (CH) catalyzes the formation of 5-hydroxymethyl-dCMP (Hm5CMP) from dCMP and methylene tetrahydrofolate (CH2THF), analogous to the reaction between dUMP and CH2THF catalyzed by thymidylate synthase (TS), an enzyme of known structure. The amino acid sequence identity between invariant TS residues and CH is at least 50%. Most of the residues which contact the dUMP and CH2THF in TS are conserved in CH. It is hypothesized that CH is homologous to TS in both structure and mechanism. The project described in this thesis tests this hypothesis. In-vitro studies on catalysis by CH variants. The roles of three residues in catalysis by CH have been tested using site-directed mutagenesis. Conversion of Cys148 to Asp, Gly or Ser decreases CH activity at least 105 fold, consistent with a nucleophilic role for Cys148 (analogous to the catalytic Cys in TS). In crystalline TS, hydrogen bonds connect O4 and N3 of bound dUMP to the side chain of an Asn; the corresponding CH residue is Asp179. Conversion of Asp179 in CH to Asn reduces kcat/KM for dCMP by 104 fold and increases kcat/KM for dUMP 60 fold, changing the nucleotide specificity of the enzyme. Other studies have shown that the specificity of TS was changed from dUMP to dCMP by conversion of the appropriate Asn to Asp. Based on the crystal structure of TS, a Glu residue (also conserved in CH) is proposed to catalyze formation of the N5 iminium ion methylene donor by protonation of N10 of CH2THF. In CH and TS, overall turnover and tritium exchange are tightly coupled. Replacement of Glu60 in CH or Glu58 in TS uncouples these catalytic steps. Conversion the Glu60/58 to Gln or Asp results in a 5-50 fold decrease in the ability to catalyze tritium exchange, consistent with an inability to catalyze formation of the N5 iminium ion, but also results in a 104-105 decrease in product formation. This suggests that Glu60/58is also involved in a step in catalysis after nucleotide and folate binding and proton removal from carbon 5 of the nucleotide. Isotope effect studies. The observed value of the α-secondary tritium inverse equilibrium isotope effect (EIE = 0.8) on formation of the complex between FdUMP, CH2THF and both wild-type CH and CH(D179N) indicates that carbon 6 of FdUMP is sp3 hybridized (tetrahedral) in the ternary complex. This is consistent with the hypothesis that that carbon 6 is bonded to Cys148 in the complex. Removal of Cys148in CH prevents complex formation with FdUMP. Lack of an observed α-secondary tritium kinetic isotope effect (KIE) for position 6 of dCMP for both enzymes suggests that the intrinsic KIE is masked by other rate-limiting steps or that rehybridization follows the first irreversible step. An observed KIE on carbon 6 of dUMP by CH(D179N) suggests the rate-limiting steps for the two nucleotide substrates is different. In-vivo studies catalysis by CH variants. In order to prevent recombination between CH deficient T4 phage and plasmid borne copies of CH variants, the gene coding for CH, gene 42, was deleted from the T4 chromosome. The T4Δ42 phage requires wild-type CH expressed from a plasmid to kill their host cell. CH variants C148G, D179N, E60Q, and E60D, all which exhibit at least 2000 fold lower activity in vitro, do not complement the T4Δ42 phage in vivo. Interchanging the functional domains of CH and TS. It is proposed that shortening the C-terminal loop seen in the structure of TS changes the solvent structure of the CH active-site such that it becomes more hydrated. Differences in the solvent structure of the active-site may account for differences in the catalytic specificity between CH and TS, respectively, hydration versus reduction. In order to test the hypothesis that these catalytic differences between TS and CH lie within the C-terminal portion of the enzyme, the N-terminus of the CH(D179N) variant was fused to the C-terminus of the wild-type TS to create a chimeric CH/TS enzyme. The chimeric enzyme was predicted to have specificity for dUMP and a active-site solvent structure similar to that for wild-type TS. However, the resulting protein cannot be overproduced to significant levels and does not have any detectable TS activity in vivo.

Les phosphoinositides (PIs) sont des phospholipides membranaires qui jouent un rôle crucial dans le contrôle de l'organisation spatio-temporelle de nombreuses voies de signalisation intracellulaire, du réarrangement du cytosquelette d'actine et du trafic de vésicules. Dans la plaquette, le métabolisme des PIs est particulièrement actif et génère, par le jeu de kinases, phosphatases et phospholipases spécifiques, des seconds messagers indispensables à l'activation plaquettaire, notamment le phosphatidylinositol 3,4,5-trisphosphate (PIP3). La première partie de la thèse concerne l'étude des différentes espèces moléculaires (composition en acides gras) des 4 grandes classes de PIs (PI, PIP, PIP2 et PIP3) dans les plaquettes humaines et de souris au repos ou lors de leur activation. Cette analyse, jamais réalisée précédemment, a été possible grâce à une technique de spectrométrie de masse (LC-MS), basée sur la méthylation avec le TMS-diazomethane des groupements phosphates des PIs. Cette étude montre une augmentation rapide et transitoire de 2 espèces moléculaires majoritaires de PIP3 lors d'une stimulation plaquettaire avec une réactivité différente des plaquettes humaines et de souris en réponse aux agonistes plaquettaires (thrombine et CRP). En utilisant des modèles murins présentant une inactivation des PI3-kinases (PI3K) dans la lignée mégacaryocytaire et des inhibiteurs spécifiques de PI3K, j'ai montré que l'isoforme PI3Kß (p110ß) de classe I est très majoritairement responsable de la production des diverses espèces moléculaires de PI(3,4,5)P3 en réponse à la thrombine ou au CRP alors que la PI3Ka (p110a) est faiblement impliquée. Les résultats montrent également une grande variété d'espèces moléculaires de PI et seulement 2 espèces moléculaires prédominantes pour les PIP, PIP2 et PIP3, aussi bien chez l'homme que chez la souris malgré des régimes alimentaires très différents. Nous montrons des différences importantes dans le métabolisme des espèces moléculaires de PI, PIP et PIP2 dans les plaquettes humaines et de souris lors de la stimulation. Dans cette étude, nous avons identifié pour la première fois des espèces moléculaires minoritaire de PIP2 mais qui augmentent de façon importante lors de la stimulation plaquettaire. Ce travail permet de dresser la première cartographie des différentes espèces moléculaires de PIs présents dans les plaquettes humaines et de souris et les modifications induites par leur activation. La deuxième partie de la thèse montre pour la première fois une localisation atypique du phosphatidylinositol 3- monophosphate (PI3P), dans le feuillet externe de la membrane plasmique plaquettaire. Je démontre que ce lipide minoritaire (environ 10% de PIP), connu pour être intracellulaire et impliqué dans le trafic vésiculaire, est également présent à la surface des plaquettes au repos. Aucun autre PI n'a pu être détecté dans le feuillet externe de la membrane plasmique plaquettaire. Ce résultat a été obtenu en utilisant différentes sondes fluorescentes se liant spécifiquement au PI3P et leurs contrôles mutées. Nous montrons que le traitement des plaquettes avec des enzymes métabolisant spécifiquement le PI3P (MTM1 et ABH) réduit significativement ce pool de PI3P. Les plaquettes de souris déficientes en PI3K de classe II et III présentent une diminution du PI3P de surface. De manière intéressante, ce pool externe de PI3P permet l'endocytose des protéines circulantes liant le PI3P, in vitro, ex vivo et in vivo. Les sondes PI3P spécifiques internalisées dans la plaquette sont stockées dans les granules a puis sécrétées lors de l'activation plaquettaire. Cette étude montre que le PI3P se comporte comme un récepteur permettant l'endocytose de protéines plasmatiques spécifiques. / Phosphoinositides (PIs) are membrane phospholipids that play a crucial role in controlling the spatiotemporal organization of many intracellular signaling pathways, actin cytoskeleton rearrangement, and vesicle trafficking. In platelet, the metabolism of PIs is highly active and generates, by the interplay of specific kinases, phosphatases and phospholipases, second messengers essential for platelet activation, in particular phosphatidylinositol 3,4,5-trisphosphate (PIP3). The first part of the thesis concerns the study of the different molecular species (fatty-acyl composition) of 4 PIs classes (PI, PIP, PIP2 and PIP3) in resting and stimulated human and mouse platelets. This analysis, never realized previously, was possible thanks to a mass spectrometry (LC-MS) technique, based on methylation of PIs phosphates groups with TMS- diazomethane. This study shows a rapid and transient increase in the 2 major molecular species of PIP3 during platelet stimulation with a different reactivity of human and mice platelets according to the used agonists (thrombin and CRP). Using mice models with selective deletion of PI3-kinases (PI3K) in the megakaryocyte lineage and specific PI3K inhibitor, I showed that the class I PI3Kß (p110ß) is the major isoform responsible for the production of the various molecular species of PIP3 in response to thrombin or CRP whereas class I PI3Ka (p110a) is weakly involved. The results also show a large variety of molecular species of PI while only 2 predominant molecular species for PIP, PIP2 and PIP3, both in humans and mice platelets despite very different diet. We show a significant difference in terms of PI, PIP and PIP2 molecular species metabolism in human and mice platelets during stimulation. In this study, we identified for the first time the presence of low-abundance molecular species of PIP2 but which increase significantly during platelet stimulation. This work constitutes the first comprehensive analysis of PIs molecular species and the changes in their actual mass during platelet stimulation. The second part of the thesis shows for the first time an atypical localization of phosphatidylinositol 3-monophosphate (PI3P), in the outer leaflet of the platelet plasma membrane. I demonstrate that this minor lipid (about 10% of PIP), known to be intracellular and involved in vesicular trafficking, is also present at the surface of resting platelet. No other PIs could be detected in the outer leaflet of the platelet plasma membrane. This result was obtained using fluorescent probes binding specifically to PI3P and their mutated controls. Treatment of platelets with PI3P specific metabolizing enzymes (MTM1 and ABH) significantly reduced this particular pool of PI3P. Class II and III PI3K deficient mouse platelets showed a decrease in surface PI3P. Interestingly, this external pool of PI3P was able to mediate endocytosis of circulating PI3P- binding proteins, in vitro, ex vivo and in vivo. Internalized specific PI3P probes were stored into platelets a-granules and could then be secreted during platelets activation. This study shows that PI3P acts as a receptor allowing endocytosis of specific plasma proteins.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2003. / Vita. Bibliography: 69-74.

Les mégacaryocytes sont des cellules de la moelle osseuse qui par un processus complexe et encore mal caractérisé, mégacaryopoïèse/thrombopoïèse, donnent naissance, in fine, aux plaquettes sanguines. La différenciation mégacaryocytaire nécessite un intense remodelage nucléaire et cytoplasmique, guidé à la fois par des facteurs intrinsèques mais aussi par des facteurs extrinsèques tel que le microenvironnement médullaire. Les plaquettes sanguines sont des acteurs essentiels du maintien de l'intégrité vasculaire. Elles sont les premiers éléments cellulaires à intervenir dans l'arrêt du saignement lors d'une blessure vasculaire par la formation d'un thrombus via des mécanismes d'adhésion, de sécrétion et d'agrégation, trois étapes majeures de l'hémostase physiologique. Dans un premier temps, mes travaux de thèse visent à déterminer le rôle inconnu de l'isoforme alpha des PI3Ks de classe II (PI3KC2a), de la PI3K de classe III (Vps34) et de leur produit, le phosphatidylinositol 3 monophosphate (PI3P), dans la production et les fonctions plaquettaires. Grâce à un modèle murin présentant une inactivation partielle de la PI3KC2a, j'ai mis en évidence son rôle clé dans la génération d'un pool basal de PI3P dans les plaquettes. L'inactivation de la PI3KC2a affecte la composition du cortex sous-membranaire plaquettaire induisant une morphologie plaquettaire anormale, une accumulation de plaquettes à deux corps appelées " barbell-shaped proplatelets ", un défaut de formation du thrombus ex vivo et un retard d'occlusion de la carotide après lésion in vivo. Ainsi, la PI3KC2a joue un rôle majeur dans le maintien de l'intégrité du squelette membranaire contrôlant la structure et la dynamique membranaire, processus critique à la production de plaquettes fonctionnelles. D'autre part, la délétion de Vps34 spécifiquement dans la lignée mégacaryocyte/plaquette se traduit par une microthrombopénie modérée associée à une migration anormale des mégacaryocytes liées à un défaut de trafic vésiculaire et une diminution du taux de PI3P. De façon intéressante, Vps34 joue aussi un rôle dans l'activation plaquettaire en régulant la production de PI3P sous stimulation, la croissance du thrombus ex vivo et les capacités thrombotiques in vivo. Le rôle de Vps34 dans la plaquette indépendamment de son rôle dans le mégacaryocyte a été confirmé via l'utilisation de nouveaux inhibiteurs spécifiques de Vps34, SAR405 et INH1, ex vivo. Vps34 est donc critique dans la régulation de la production plaquettaire par les mégacaryocytes ainsi que dans l'activation plaquettaire. Dans un deuxième temps, je me suis intéressé à l'impact du microenvironnement médullaire sur la mégacaryopoïèse, et plus spécifiquement sur la communication entre adipocytes médullaires et progéniteurs hématopoïétiques lors de leur différenciation en mégacaryocytes. Grace à un système de coculture in vitro, j'ai montré que les adipocytes améliorent la différenciation mégacaryocytaire via un transfert direct de lipides, dans un but non-énergétique. Dans un contexte d'obésité, nous observons, in vivo, associée à une adiposité médullaire augmentée une maturation mégacaryocytaire exacerbée, une production et une demi-vie plaquettaire défectueuses ayant pour conséquence une macrothrombopénie. Ainsi, le microenvironnement médullaire et plus particulièrement l'adipocyte impacte directement sur la mégacaryopoïèse et la production plaquettaire. En conclusion, ces travaux de thèse contribuent à caractériser les mécanismes de production et de fonction plaquettaire régulés par des facteurs intrinsèques tels que le PI3KC2a et Vps34, ainsi que par des facteurs extrinsèques tels que l'adipocyte médullaire. / Megakaryopoiesis is a highly specialised and complex process occurring in the bone marrow, by which megakaryocytes give rise to de novo circulating blood platelets. Megakaryocyte differentiation implies cytoplasmic and nuclear rearrangements regulated by intrinsic as well as extrinsic factors such as bone marrow microenvironment. Platelets play a critical role in preventing blood loss after vascular injury by orchestrating clot formation through mechanisms of adhesion, secretion and aggregation. These mechanisms are the three major steps of physiological haemostasis leading to the maintenance of vascular integrity. Firstly, my thesis work focused on characterizing the role of class II PI3K alpha isoform (PI3KC2a), class III PI3K (Vps34) and their common product the phosphatidylinositol 3 monophosphate (PI3P) in platelet production and function. Using a unique mouse model partially inactivated for PI3KC2a, I highlighted its key role in the production of a basal PI3P housekeeping pool in platelets. PI3KC2a partial inactivation affects platelet membrane skeleton composition leading to an abnormal platelet morphology, an enrichment of platelet with two cell bodies recently called "barbell-shaped proplatelets", an ex vivo defective thrombus formation and an in vivo delayed carotid occlusion following injury. Thus, PI3KC2a plays a major role in membrane structure and dynamics by maintaining membrane skeleton integrity, which is crucial for functional platelet production. On the other hand, Vps34 specific deletion in megakaryocyte/platelet lineage induced mild microthombopenia correlated to an abnormal megakaryocyte migration linked to an affected PI3P production as well as vesicular trafficking in megakaryocytes. In platelets, Vps34 plays a role in their activation by regulating PI3P production under stimulation, ex vivo thrombus growth and in vivo thrombotic capacity. Vps34 role in platelet independently from its role in megakaryocyte was confirmed using two recently developed inhibitors, SAR405 and INH1, which reproduced ex vivo thrombus growth defects. Therefore, Vps34 is critical for platelet production by megakaryocyte as well as platelet activation. Secondly, I studied the impact of bone marrow microenvironment on megakaryopoiesis and more specifically the crosstalk between medullar adipocytes and hematopoietic progenitors differentiating towards the megakaryocyte lineage. Using an in vitro coculture assay, I demonstrated that adipocytes enhanced megakaryocyte differentiation through a direct lipid transfer, in a non-energetic aim. In the context of obesity, increased marrow adipocity is associated to enhanced megakaryocyte differentiation and defective platelet production and lifespan leading to macrothrombopenia. Thus, bone marrow microenvironment through adipocytes impact directly on megakaryopoiesis and platelet production. Altogether my thesis work contributes to better understand platelet production and function, mechanisms regulated by intrinsic factors such as PI3KC2a and Vps34 as well as extrinsic factors like medullar adipocytes.

The role of cyclic AMP and a differentiation-indueing factor (DIF) in the differentiation of stalk cells was investigated in the cellular slime mold Dictyostelium discoideum. In this organism, starvation triggers the aggregation of amoebae into multicellular masses within which a simple, well-regulated pattern of partially differentiated cells is formed and which ultimately form fruiting bodies comprised of spore and stalk cells. In a monolayer system at low cell densities, stalk cell formation is dependent on the presence of both cyclic AMP and DIF. Both factors act within a short time of each other, induction by cyclic AMP preceding induction by DIF, beginning between 8 to 10 hours of incubation in monolayers, and progressively committing an increasing proportion of the cells in monolayer to form stalk cells. The relative effectiveness of analogues of cyclic AMP to induce stalk cell formation in monolayers indicates that the well-characterized cell surface cyclic AMP receptor most probably mediates the action of cyclic AMP. Although this receptor appears early during aggregation, it does not become activated until later during development in vivo, probably because the cyclic AMP concentrations within developing cell masses must build up to levels higher than those in aggregation streams. The finding that caffeine inhibits stalk cell formation in low density monolayers and that the permeable analogue 8-Bromo-cyclic AMP can partially reverse this inhibition suggests that activation of this receptor leads to an increase in internal cyclic AMP levels as one of the steps in stalk cell differentiation. The finding that the expression in low density monolayers of AP IV, a cell-type non-specific isozyme of acid phosphatase, was cyclic AMP-dependent is consistent with the view that cyclic AMP induces non-specific postaggregative gene expression during development in vivo. The findings that the expression of pre-stalk arid stalk cell specific antigens and of the pre-stalk cell specific isozyme AP II was DIF-dependent provide good evidence for the idea that both pre-stalk and stalk cell formation are induced by DIF. The fact that isolated pre-stalk cells require DIF for stalk cell formation in low density monolayers further supports this idea. Whereas cells independent of DIF for stalk cell formation in monolayers appear immediately after cyclic AMP-independent cells during differentiation in low density monolayers, DIF-independent cells appear considerably later during development in vivo. This evidence and the fact that developing cell masses contain elevated levels of DIF lead to the postulate that the factor(s) which triggers the formation of fruiting bodies also controls the pre-stalk to stalk cell conversion. / Science, Faculty of / Botany, Department of / Zoology, Department of / Graduate

Yes / Ribonucleoside monophosphate (rNMP) incorporation in genomic DNA poses a significant threat to genomic integrity. In addition to repair, DNA damage tolerance mechanisms ensure replication progression upon encountering unrepaired lesions. One player in the tolerance mechanism is Rad5, which is an E3 ubiquitin ligase and helicase. Here, we report a new role for yeast Rad5 in tolerating rNMP incorporation, in the absence of the bona fide ribonucleotide excision repair pathway via RNase H2. This role of Rad5 is further highlighted after replication stress induced by hydroxyurea or by increasing rNMP genomic burden using a mutant DNA polymerase (Pol ε - Pol2-M644G). We further demonstrate the importance of the ATPase and ubiquitin ligase domains of Rad5 in rNMP tolerance. These findings suggest a similar role for the human Rad5 homologues helicase-like transcription factor (HLTF) and SNF2 Histone Linker PHD RING Helicase (SHPRH) in rNMP tolerance, which may impact the response of cancer cells to replication stress-inducing therapeutics.

La découverte de nouvelles substances thérapeutiques nécessite la synthèse de série de molécules soumises au criblage biologique sur une cible donnée. Ce projet de recherche a pour objectif de développer des inhibiteurs de nucléosides monophosphate kinases (NMPK) en se basant sur le concept de chimie dynamique combinatoire in situ. La synthèse de ces molécules a nécessité l’association via des fonctions réactives d’un analogue d’accepteur de phosphate et d’un mime d’ATP donneur de phosphate. La mise au point de ce dernier a fait l’objet de ce travail de thèse et a été orientée vers la pharmacomodulation d’un hétérocycle azoté et soufré: la 2-thiohydantoïne. La synthèse de ce composé a été réalisée par la méthode de Schlack-Kumpf et par celle d’Edman provenant de techniques d’analyses peptidiques. Ces deux voies ont été exploitées pour étudier la réactivité et la fonctionnalisation sélective de cet hétérocycle notamment par des couplages de type Suzuki. La réaction de Vilsmeier-Haack-Arnold a par la suite constitué l’étape clé permettant de transformer un cycle 2-thiohydantoïne en un cycle de type imidazole qui a pu être fonctionnalisé en diverses positions. La synthèse de dérivés 2-thiohydantoïne et imidazole diversement substitués par des groupements utiles, au couplage in situ avec les analogues d’accepteur de phosphate ainsi qu’à l’affinité enzymatique a permis l’accès à une bibliothèque de molécules. Des tests biologiques ont permis d’évaluer leur affinité vis-à-vis de plusieurs NMPK ainsi que leur cytotoxicité sur cellules cancéreuses ; cet ensemble de résultats permettant de trouver les déterminants nécessaires à l’activité biologique. / New therapeutical compounds determination requires the formation of a library of molecules and their screening on specific biological targets. The aim of this project was to design new inhibitors targeting nucléoside monophosphate kinases (NMPK) based on in situ dynamic combinatorial chemistry. These molecules were synthesized by ligation between analogues of phosphate acceptors and donors on which reactive functions were introduced. The topic of this PhD was to develop the ATP mimetics using chemical transformation and pharmacomodulation of a small heterocycle: 2-thiohydantoin. Its synthesis was achieved using the Schlack-Kumpf and the Edman methods initially develop for peptidic analysis. These two pathways have been explored in order to study the reactivities and the selective functionalizations of the heterocycle allowing for example Suzuki cross coupling reactions. Furthermore we used the Vilsmeier-Haack-Arnold reaction as a key step to the formation of a highly substituted imidazole ring directly from a 2-thiohydantoin. The synthesis of 2-thiohydantoin and imidazole derivatives, on which reactive groups for the in situ coupling reactions and the enzymatic affinity have been introduced, leads to a library of molecules. Their affinity toward to ATP donor site of NMPK and their toxicity on cancer cells were evaluated by biological tests.