La phosphorylation/déphosphorylation des protéines est une modification clé dans les mécanismes qui contrôlent les évènements mitotiques.Classiquement, l’entrée en mitose requiert l’activation de Cdk1. Pour se faire, les phosphorylations inhibitrices sur Cdk1 par Myt1 et Wee1 doivent être éliminées par Cdc25. Le complexe Cdk1-Cycline B (MPF) est ainsi actif, les kinases inhibitrices inactivées.Dernièrement, une nouvelle protéine kinase clé pour l’entrée en mitose a été mise en évidence : Greatwall (Gwl). Les récents résultats publiés par notre équipe montrent que Gwl permet l’entrée et le maintien en mitose en inhibant l’activité de la phosphatase PP2A, la phosphatase responsable de la déphosphorylation des substrats de la protéine kinase Cdk1-Cycline B, via son substrat Arpp19. Gwl phosphoryle Arpp19 sur la sérine 71 lui conférant ainsi la capacité d’inhiber l’activité de la phosphatase PP2A.Une étude sur les modifications post traductionnelles d’Arpp19 a été initiée dans l’équipe et met en évidence plusieurs sites de phosphorylation : <br>• La sérine 71, site de phosphorylation par Gwl <br>• La sérine 28, dont la phosphorylation est attribuée à Cdk1 (vérifié in vitro) <br>• La sérine 113, site de phosphorylation par pKA <br>Ce projet de thèse s’inscrit dans la suite logique du travail déjà effectué dans l’équipe et a pour objectif de caractériser les modifications post traductionnelles d’Arpp19, leurs rôles dans la progression mitotique, leurs incidences sur la liaison et l’inhibition de la cible d’Arpp19, PP2A.Cette partie du projet repose sur la synthèse de mutants d’Arpp19Xe, mutants phosphomimétiques d’une part (sérine transformée en acide aspartique par mutagenèse dirigée) ou mutants dont la phosphorylation est impossible (sérine en alanine). Ces mutants nous ont permis de travailler sur l’impact de ces différentes phosphorylations dans l’extrait d’œufs de Xénope.Ce projet s’attache également à mettre en lumière l’ensemble de la voie de signalisation aboutissant aux différentes modifications post traductionnelles d’Arpp19, leurs chronologies au cours du cycle et ainsi identifier les protéines effectrices de ces phosphorylations sur Arpp19 qui sont autant de leviers potentiels sur lesquels les thérapies anti-tumorales pourraient s’appuyer. / Proteins phosphorylation and dephosphorylation are key post translational modifications controlling mitotic events.Traditionally, mitotic entry requires Cdk1 activation. To allow this to occur, inhibitory phosphorylations on Cdk1 by Myt1 and Wee1 kinases must be removed by phosphatase Cdc25. Thus, the Cdk1-Cyclin B complex, also called MPF (Mitotic Promoting Factor), is active and inhibitory kinases inactivated.Along this canonic scheme, another key kinase has been shown to play a critical role: the Greatwall (Gwl) kinase also called MAST-L for MAST like. Results published by our team show that in Xenopus laevis, Gwl allows entry and maintains mitosis by inhibiting the activity of the phosphatase responsible for dephosphorylation of Cdk1/Cycline B substrates: PP2A. This activity is driven by Gwl target: Arpp19. Gwl phosphorylates Arpp19 on its 71st residue turning it into a potent inhibitor of PP2A.A study of Arpp19 post translational modifications of Arpp19 has been initiated in the team which will allow the further study of several phosphosites: <br>• Serine 71, Gwl phosphosite, the best documented site. <br>• Serine 28, shown in vitro to be a Cdk1-CycB phosphosite. <br>• Serine 113, assigned to PKA. <br>This thesis project joins logically after the work already made in the team and has for objective to characterize the post translational modifications of Arpp19, their roles in mitotic progress, their incidences on binding and inhibition of Arpp19’s target, PP2A.This part of the project relies on mutants' synthesis of Arpp19Xe, phosphomimetics’ mutants on one hand (serine transformed into aspartic acid by mutagenesis) or mutants unable to be phosphorylated (serine into alanine). These mutants allowed us to work on the impact of these various phosphorylations in Xenopus eggs extracts.This project also attempts to highlight the whole signalization pathway ending in the various post translational modifications of Arpp19, their timelines during the cycle and thus to identify effector proteins of these phosphorylations on Arpp19 which are as much as potential levers on which can serve as targets for cancer therapy.

Le diabète de type 2 se caractérise par une résistance à l’insuline dans les tissus périphériques, une déficience de sécrétion d’insuline et une augmentation de la production endogène de glucose. Notre but a été de démontrer le rôle spécifique de la production hépatique de glucose dans le développement du diabète. Nous avons développé un modèle de souris invalidées pour le gène codant pour la sous-unité catalytique de la G6Pase, enzyme clé de la production de glucose, spécifiquement dans le foie. Sous alimentation diabétogène, les souris transgéniques résistent au développement de l’hyperglycémie et l’hyperinsulinémie, et présentent une amélioration de la sensibilité à l’insuline et une augmentation du captage périphérique de glucose. Ces souris résistent également à l’obésité induite par ce régime déséquilibré, en liaison avec l’augmentation de la dépense énergétique, associée à l’induction des médiateurs de la thermogenèse dans les tissus adipeux brun et blanc, et au remodelage du muscle squelettique vers un phénotype oxydative. La délétion de la G6PC hépatique chez des souris rendues obèses et diabétiques résulte en une amélioration spectaculaire et rapide du métabolisme glucidique, et une stabilisation de la masse corporelle des souris obèses, associée à une induction des gènes du métabolisme oxydative dans les tissus périphériques.Ces effets bénéfiques pourraient être dus à l’augmentation de la sécrétion de facteurs hépatiques circulants connus pour réguler le métabolisme énergétique et glucidique dans les tissus périphériques. Ces travaux démontrent le rôle délétère de la production hépatique de glucose dans le développement du diabète et de l’obésité / Type 2 diabetes is characterized by insulin resistance of glucose uptake by peripheral tissues, insulin secretion deficiency and increased endogenous glucose production. Our aim is to demonstrate the specific role of hepatic glucose production in triggering insulin resistance and diabetes. For that, we developed an inducible and liver-specific knock-out mouse model for the gene encoding the catalytic subunit of G6Pase, a key enzyme of glucose production. When fed a high fat/high sucrose diet, transgenic mice resisted to the development of fasting hyperglycemia and hyperinsulinemia, and even showed enhanced insulin sensitivity and glucose uptake in peripheral tissues. These mice are also resistant to diet induced obesity, due to the induction of basal metabolism, associated with increased brown and white adipose tissue thermogenesis machinery and remodeling of skeletal muscle towards a more oxidative phenotype. When liver G6pc deletion was realized in obese and diabetic mice, this resulted in a spectacular and early amelioration of glucose metabolism compared to that before liver G6pc deletion, and to stabilization of body mass of obese mice, which was associated with induction of oxidative genes in peripheral tissues. These beneficial effects could be explained by the secretion of hepatic circulating hormones known to control glucose and energy metabolism in peripheral tissues. This work underlines the deleterious role of hepatic glucose production in the development of obesity and diabetes, and sets the liver as a master-switch in the regulation of whole-body glucose and energy metabolism

The kinase interaction motif (KIM) family of protein-tyrosine phosphatases (PTPs) includes hematopoietic protein-tyrosine phosphatase (HePTP), striatal-enriched protein-tyrosine phosphatase (STEP), and protein-tyrosine phosphatase receptor type R (PTPRR). KIM-PTPs bind and dephosphorylate mitogen-activated protein kinases (MAPKs) and thereby critically modulate cell proliferation and differentiation. PTP activity can readily be diminished by reactive oxygen species (ROS), e.g. H2O2, which oxidize the catalytically indispensable active-site cysteine. This initial oxidation generates an unstable sulfenic acid intermediate that is quickly converted into either a sulfinic/sulfonic acid (catalytically dead and irreversible inactivation) or a stable sulfenamide or disulfide bond intermediate (reversible inactivation). Critically, our understanding of ROS-mediated PTP oxidation is not yet sufficient to predict the molecular responses of PTPs to oxidative stress. However, identifying distinct responses will enable novel routes for PTP-selective drug design, important for managing diseases such as cancer and Alzheimer's disease. Therefore, we performed a detailed biochemical and molecular study of all KIM-PTP family members to determine their H2O2 oxidation profiles and identify their reversible inactivation mechanism(s). We show that despite having nearly identical 3D structures and sequences, each KIM-PTP family member has a unique oxidation profile. Furthermore, we also show that whereas STEP and PTPRR stabilize their reversibly oxidized state by forming an intramolecular disulfide bond, HePTP uses an unexpected mechanism, namely, formation of a reversible intermolecular disulfide bond. In summary, despite being closely related, KIM-PTPs significantly differ in oxidation profiles. These findings highlight that oxidation protection is critical when analyzing PTPs, for example, in drug screening.

Die leukozytäre alkalische Phosphatase (leukoAP) wird in der Humanmedizin zur Charakteristik stabkerniger neutrophiler Granulozyten und damit der Feststellung leukozytärer Reaktionen im Sinne der Linksverschiebung genutzt. Des weiteren dient die quantitative Erfassung der leukoAP der Funktionsbeschreibung dieser Zellart. Im Vergleich dazu liegen bisher beim Rind nur wenige Untersuchungen vor. Ziel dieser Arbeit war es deshalb, entsprechende Grundlagen für diese Tierart zu erarbeiten, d.h., die Bestimmungsmethodik an Rindergranulozyten zu adaptieren, Referenzwerte zu definieren sowie klinische Bezüge bei ausgewählten Krankheiten zu prüfen. Die Untersuchungen wurden an gesunden weiblichen Kälbern im Alter von drei bis vier (n = 13), sechs bis acht (n = 13) und 12 bis 14 Wochen (n = 11), weiblichen Jugrindern im Alter von sechs (n = 9) und zwölf Monaten (n = 10) sowie dreijährigen Kühen in der Trockenstehperiode (n = 11), fünf bis sieben Wochen (n = 10) sowie 12 bis 14 Wochen post partum (n = 6) durchgeführt. Außerdem wurden Kühe mit Dislocatio abomasi (n = 8) und mit weiteren Erkrankungen (n = 8) analysiert. Zur Analytik der leukoAP wurde ein durchflußzytometrisches Verfahren für Rinder adaptiert, das eine schnellere und genauere Arbeit als das herkömmliche Verfahren der leukoAP-Index-Bestimmung ermöglicht. Weiterhin wurden die Aktivitäten der AP und dessen Knochenisoenzyms im Blutserum sowie das Leukogramm bestimmt. Die Bestimmung der leukoAP ist auf der Basis der Diazoniumfärbung auch beim Rind durchflußzytometrisch mit großer Präzision und hohem Probendurchsatz möglich. Die leukoAP-Aktivitäten (relative Fluoreszenzaktivitäten) zeigen eine Altersabhängigkeit. Sie bewegen sich bei drei bis vier Wochen alten gesunden Kälbern zwischen 2,2 bis 5,7 (Mittelwert: 3,8), sinken kontinuierlich bis in den Bereich 1,9 bis 4,3 (Mittelwert: 2,8) bei 12 bis 14 Wochen alten Kälbern ab (p£0,05), um bis auf 2,6 bis 5,5 (Mittelwert: 4,2) bei einjährigen Jungrindern wieder signifikant (p£0,05) anzusteigen. Bei gesunden, trockenstehenden dreijährigen Kühen sowie Kühen 12 bis 14 Wochen post partum schwanken die leukoAP-Aktivitäten zwischen 1,2 bis 9,7 (Mittelwerte bei 3,3 bzw. 3,6). Im Zeitraum fünf bis sieben Wochen post partum ist die leukoAP-Aktivität mit 2,1 bis 8,2 (Mittelwert: 5,3) signifikant (p£0,05) höher. Bei der Gesamtheit der untersuchten gesunden Rinder korreliert die leukoAP weder mit der Zahl der Leukozyten (gesamt), der stabkernigen neutrophilen Granulozyten, der segmentkernigen neutrophilen Granulozyten, der Monozyten, noch mit der Zahl der Lymphozyten systematisch gesichert. Andererseits sind Beziehungen zu diesen Parametern für einzelne Alters- und Laktationsgruppen nachweisbar. Die leukoAP korreliert weder mit der Gesamtaktivität noch mit dem Knochenisoenzym der AP im Serum. Kühe haben einen Tag nach Reposition einer Dislocatio abomasi leukoAP-Aktivitäten von 2,4 bis 7,4 (1. und 3. Quartil). Innerhalb von drei Tagen post repositionem steigt die leukoAP-Aktivität in den Bereich 5,1 bis 11,5 signifikant an, während die Leukozytenzahl sinkt (p£0,05). Bei Kühen mit linksseitiger Dislocatio abomasi verlaufen die leukoAP-Aktivität sowie der Anteil stabkerniger neutrophiler Granulozyten postoperativ parallel. Bei Kühen mit entzündlichen Erkrankungen (Klauensohlengeschwüre, Retentio secundinarum, Pneumonie, Ekzem) sind z.T. stark erhöhte leukoAP-Aktivitäten feststellbar. Sie gehen tendenziell mit einem Anstieg der stabkernigen neutrophilen Granulozyten sowie mit Leukozytose einher, im Einzelfall besteht jedoch nicht immer dieser Trend. Die Bestimmung der leukoAP ist nur bei gegebenen technischen Voraussetzungen eine auch bei Rindern einfach durchführbare Methode zur Charakteristik der neutrophilen Granulozyten mit altersabhängigen Differenzen. Bei Dislocatio abomasi besteht eine Reduzierung der leukoAP-Aktivität, die post repositionem jedoch erheblich zunimmt. / Leukocyte alkaline phosphatase (leukoAP) is used in the human medicine to characterize unsegmented neutrophile granulocytes and thereby to diagnose leukocyte reactions in terms of a leftward shift. Furthermore, the quantitative capture of leukoAP serves as a functional description of this cell type. To our knowledge, only little investigation has been done on cattle. Therefore, this dissertation aimed to acquire an equal basis for this species, i.e. to adapt the methodology of determination, to define reference values, and to examine relevant relationship for selected diseases. Healthy female calves aged between three and four (n = 13), six and eight (n = 13), and between twelve and fourteen weeks (n = 11), cows aged six (n = 9) and twelve months (n = 10), three-year-old cows ante partum (n=11), and ten cows between five and seven as well as six cows between twelve and fourteen weeks post-partum were used in the study. In addition, eight cows with abomasal dislocation and another eight with other complications were examined. For analyzing leukoAP we adapted flow cytometry for cattle which delivers faster and more exact determination than the conventional method of leukoAP index determination. Moreover, the activity of alkaline phosphatase (AP) in blood serum and its iso-enzymes in the bone as well as leukogrammes were determined. The flow cytometrical determination of leukoAP activity was based upon the diazonium staining technique which also provides an accurate measurement in a large number of samples for cattle. The leukoAP activities (given as a relative fluorescence activity) showed age dependency: they ranged between 2.2 and 5.7 (mean value 3.8) in calves of 3 to 4 weeks in age, and decreased continuously and significantly (p£0,05) to 1.4 - 4.3 (mean value 2.8) in calves between 12 and 14 weeks of age, but increased again significantly (p£0,05) ranging between 2.6 - 5.5 (mean value 4.2) in one-year-old cattle. In healthy cows, ante partum and 12 to 14 weeks post-partum, differed the leukoAP activity between 1.2 and 9.7 (mean value 3.3 and 3.6, respectively), whereas in cows 5-7 weeks post-partum the leukoAP activity was significantly greater reaching 2.1 to 8.2 (mean value 5.3, p£0,05). For healthy cattle, leukoAP correlated neither with the total number of leukocytes, unsegmented neutrophile granulocytes, monocytes nor with the number of lymphocytes. In addition, leukoAP correlated neither total activity nor with serum AP and its iso-enzymes in the bone. One day after replacing the abomasal dislocation (between 1st and 3rd quartile) cows showed a significant decrease in leukoAP activity (2.4 - 7.4) vs. healthy cows after the same period of time post-partum. This parameter increased, however, significantly three days post-operative (5.1-11.5), whereas the number leukocytes decreased (p£0,05). In cows with left side abomasal dislocation, leukoAP activity and the number of unsegmented neutrophile granulocytes post-operative developed concomitantly. We found enhanced leukoAP activity in cows with different inflammatory disorders such as sole ulcer, placenta retention, pneumonia and eczema which provably (p£0,05) corresponded with an increased number of unsegmented neutrophile granulocytes and partly leukocytosis. However, for isolated cases this trend did not always show. Determining the leukoAP is, hence, a method for characterizing neutrophile granulocytes depending on age differences and is only practible for cattle if the required technical conditions are given. During abomasal dislocation, leukoAP activity decreased, but increased considerably after post-operative replacement.

Tableau d'honneur de la Faculté des études supérieures et postdoctorales, 2005-2006 / Le neutrophile est spécialisé dans la chimiotaxie et la phagocytose. Ces deux phénomènes sont accompagnés d'une accumulation de phosphatidylinositol(3,4,5)triphosphate. Ce dernier est formé par les phosphatidylinositols 3-kinases. Leur activation et la formation de phosphatidylinositol(3,4,5)triphosphate sont bien caractérisés dans le neutrophile. La régulation du niveau de phosphatidylinositol(3,4,5)triphosphate par les lipide phosphatases est peu étudiée. Nous avons examiné le rôle des lipide phosphatases SHIP1 et PTEN suite à la stimulation du CD32a, un FcyR, dans la régulation du niveau de phosphatidylinositol(3,4,5)triphosphate. Ce dernier augmente suite à la stimulation du CD32a. La localisation cellulaire et la phosphorylation de SHIP1, mais pas de PTEN, sont modifiées en réponse à la stimulation du CD32a. Ces événements seraient dépendants des Src kinases. Par contre, ils seraient indépendants de l'activation des phosphatidylinositol 3-kinases. SHIP1, et non PTEN, pourrait donc être impliquée dans la régulation du phosphatidylinositol(3,4,5)triphosphate formé suite à la stimulation du CD32a dans le neutrophile humain.

Résumé : Les podocytes, cellules épithéliales rénales, sont nécessaires au maintien de la structure et de la fonction de filtration des glomérules rénaux. La dédifférenciation et l’apoptose des podocytes sont des évènements précoces de la néphropathie diabétique. Des études ont rapporté que l’insuline est nécessaire à la survie des podocytes puisque la délétion du récepteur à l’insuline dans les podocytes de souris entraîne une pathologie glomérulaire semblable à la néphropathie. D’autres études ont montré que la protéine tyrosine phosphatase Src homology-2 domain-containing phosphatase-1 (SHP-1) inhibe les voies de signalisation de l’insuline au niveau du foie et du muscle en déphosphorylant la sous-unité bêta du récepteur à l’insuline (IRβ) et la kinase Phosphatidylinositide 3-kinase (PI3K). Il a récemment été démontré que l’expression de SHP-1 est élevée dans les cortex rénaux de souris diabétiques. Nous avons donc émis l’hypothèse que l’expression de SHP-1 induite par l’hyperglycémie altère les actions de l’insuline dans les podocytes. Nous avons premièrement utilisé un modèle in vivo de souris diabétiques de type 1 (Ins2+/C96Y; Akita). Comparées aux souris contrôles (Ins2+/+), les souris Akita présentaient une apoptose élevée des podocytes ainsi qu’une perte des pédicelles. La phosphorylation de la protéine kinase B (Akt) et de Extracellular signal-regulated kinase 1/2 (ERK1/2), suite à une injection systémique d’insuline, était également significativement diminuée dans les cortex rénaux des souris Akita. Cette diminution correspondant à une résistance à l’insuline corrélait avec une augmentation de deux fois de l’expression de SHP-1 dans les glomérules. Nous avons ensuite utilisé une lignée immortalisée de podocytes murins en culture et avons observé que l’exposition à des concentrations élevées de glucose (HG; 25 mM) pendant 96 h, entraînait l’augmentation de l’expression de marqueurs apoptotiques et de l’activité enzymatique de caspase-3/7 en comparaison aux concentrations normales de glucose (NG; 5,6 mM). L’exposition en HG a augmenté l’expression de l’ARNm et protéique de SHP-1, en plus de réduire la signalisation de l’insuline dans les podocytes. La surexpression de la forme dominante-négative de SHP-1 dans les podocytes a permis de renverser les effets de HG et de restaurer les actions de l’insuline. Finalement, l’augmentation de l’expression de SHP-1, tant in vivo qu’in vitro, a été directement corrélée à son association avec IRβ et à la diminution de la phosphorylation de IRβ, Akt et ERK1/2 suite à une stimulation à l’insuline. En conclusion, nous avons montré que l’expression élevée de SHP-1 dans les glomérules cause une résistance à l’insuline et la mort des podocytes contribuant ainsi à la néphropathie diabétique. // Abstract : Podocytes are epithelial renal cells required to preserve glomerular structure and filtration. Their dedifferentiation and apoptosis are early events of diabetic nephropathy progression. Previous studies have shown that insulin action is critical for podocyte survival since deletion of its receptor lead to a glomerular pathology similar to nephropathy. It has also been demonstrated that Src homology-2 domain-containing phosphatase-1 (SHP-1), a protein tyrosine phosphatase, inhibits insulin signaling pathway in liver and muscle by dephosphorylating tyrosine residues on insulin receptor beta-subunit (IRβ) and the Phosphatidylinositide 3-kinase (PI3K). A recent study concluded that SHP-1 is elevated in kidney cortex of type 1 diabetic mice. We hypothesized that hyperglycemia-induced SHP-1 expression may affect insulin actions in podocytes. To confirm this hypothesis, we used type 1 diabetic Akita mice (Ins2+/C96Y). Compared to control littermate mice (Ins2+/+), Akita mice developed elevated podocyte foot process effacement and podocyte apoptosis. In contrast to control mice, insulin-stimulated protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation was remarkably reduced in renal podocytes of Akita mice. This phosphorylation diminution associated to a renal insulin resistance was correlated with a two-fold increase of SHP-1 expression in the glomeruli. We then used cultured murine podocytes cell line to confirm our in vivo results. Podocytes exposed to high glucose concentration (HG; 25 mM) for 96 h exhibited high levels of apoptotic markers and caspase-3/7 enzymatic activity as compared to normal glucose concentration (NG; 5,6 mM). HG exposure raised mRNA and protein levels of SHP-1 and reduced the insulin-signaling pathway in podocytes. Overexpression of dominant-negative SHP-1 in podocytes prevented HG effects and restored insulin actions. Finally, elevated SHP-1 expression induced by high glucose levels was directly correlated to an increased association with insulin receptor-β subunit (IRβ) in vitro and in vivo. This association is therefore leading to the reduction of both IRβ phosphorylation and insulin-stimulated Akt and ERK phosphorylation. In conclusion, our results showed that high levels of SHP-1 in glomeruli cause insulin resistance and podocyte loss, thereby contributing to diabetic nephropathy.

BACKGROUND: Prostate cancer (PCa) is the most commonly diagnosed solid tumor and the second leading cancer death in the United States, and also one of the major cancer-related deaths in Chinese. Androgen deprivation therapy (ADT) is the first line treatment for metastatic PCa. PCa ultimately relapses with subsequent ADT treatment failure and becomes castrate-resistant (CR). It is important to develop effective therapies with a surrogate marker towards CR PCa. METHOD: Histone deacetylase (HDAC) inhibitors were examined to determine their effects in androgen receptor (AR)/ cellular prostatic acid phosphatase (cPAcP)-positive PCa cells, including LNCaP C-33, C-81, C4-2 and C4-2B and MDA PCa2b androgen-sensitive and androgen-independent cells, and AR/cPAcP-negative PCa cells, including PC-3 and DU 145 cells. Cell growth was determined by cell number counting. Western blot analyses were carried out to determine AR, cPAcP and PSA protein levels. RESULTS: cPAcP protein level was increased by HDAC inhibitor treatment. Valproic acid, a HDAC inhibitor, suppressed the growth of AR/cPAcP-positive PCa cells by over 50% in steroid-reduced conditions, higher than on AR/cPAcP-negative PCa cells. Further, HDAC inhibitor pretreatments increased androgen responsiveness as demonstrated by PSA protein level quantitation. CONCLUSION: Our results clearly demonstrate that HDAC inhibitors can induce cPAcP protein level, increase androgen responsiveness, and exhibit higher inhibitory activities on AR/cPAcP-positive PCa cells than on AR/cPAcP-negative PCa cells. Upon HDAC inhibitor pretreatment, PSA level was greatly elevated by androgens. This data indicates the potential clinical importance of cPAcP serving as a useful biomarker in the identification of PCa patient sub-population suitable for HDAC inhibitor treatment.

The Twin arginine translocase (Tat) system catalyzes movement of folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plant chloroplasts. This transport process requires energy in the form of the transmembrane proton motive force (PMF). The Tat system can be studied in vitro using inner membrane vesicles (IMVs) from E. coli overproducing the Tat components, TatA, TatB and TatC. However, the transport efficiencies of current in vitro Tat transport assays are low. In this work, current in vitro Tat transport assays were compared and parameters that affect transport efficiencies were identified. Mild French press treatment of IMVs resulted in larger IMVs with higher transport efficiencies. Chloride ions were shown to inhibit Tat transport in vitro. Generation of a PMF by the activity of ATP synthase gave higher transport efficiencies than generating a PMF by NADH respiration. This understanding was applied to develop an optimized in vitro Tat transport assay that showed a higher transport efficiency than currently published methods. Fluorescently labelled Tat substrates were developed to allow quantitative analysis of Tat transport. The transport of the purified native Tat substrate, CueO into IMVs was characterized using the optimized in vitro Tat transport assay. It was shown that the proton concentration (ΔpH) component of the PMF was sufficient to support Tat transport in vitro. It was observed that transport of CueO ceased in a time-dependent manner in the in vitro Tat transport assays. This loss of transport efficiency could be due, at least in part, to the presence of a PMF since transport efficiency was reduced when IMVs were pre-energized. Substrates for future in vitro single molecule fluorescence microscopy studies of the Tat transport were developed in this work. One of the substrates is fluorescently labelled CueO. The second substrate is the native Tat substrate alkaline phosphatase PhoX from Vibrio fischeri which was able to cleave the fluorogenic compound AttoPhos® and can thus be used as an enzymatic reporter of Tat transport. The structure of a native Tat substrate from Pseudomonas fluorescens, PhoX, was solved by X-ray crystallography at a resolution of 1.4Å. PhoX is a monomeric six blade β propeller with two α-helical bundle subdomains. PhoX was shown to have optimum activity at pH8.0. PhoX has a novel catalytic site which requires two Fe³⁺ (including a Cys-coordinated Fe³⁺) and three Ca²⁺ as cofactors. Mutagenesis studies showed that all the metal ions are required for the integrity of the active site. Co-crystallization of PhoX with vanadate, an inhibitor of PhoX which mimics the transition state, showed that hydrolysis of phosphomonoesters does not involve formation of a covalent phosphoenzyme intermediate. Instead, dephosphorylation of substrates is proposed to occur via a SN2 reaction with OH- as the attacking nucleophile.

<p>Apoptosis is a programmed form of cell death, essential for maintaining tissue homeostasis and eliminating dysfunctional cells. The process of apoptosis is executed by a family of cysteine proteases called caspases. High levels of metabolic activity confer resistance to apoptosis. Caspase-2, an apoptotic initiator, can be suppressed by high levels of nutrient flux through the pentose phosphate pathway (PPP). This metabolic suppression of caspase-2 is exerted via the inhibitory phosphorylation of S135 on the caspase-2 prodomain by activated Ca2+/Calmodulin-dependent protein kinase II (CaMKII). However, it was unclear how CaMKII activity is regulated by nutrient flux.</p><p>After investigating how nutrient flux leads to activation of CaMKII, a recent study reported that coenzyme A (CoA) can directly bind to and activate CaMKII. However, by performing mass spectrometry (MS) analysis of CaMKII, and other biochemical assays, including gel filtration assays, immuno-precipitation assays, immuno-depletion assays, and in vitro kinase assays, in the Xenopus egg extract system, our studies show that the complete nutrient-driven CaMKII activation requires the additional release of a "brake" through the dephosphorylation of CaMKII at novel sites (T393/S395). Furthermore, this metabolically-stimulated dephosphorylation of CaMKII is mediated by the metabolic activation of protein phosphatase 2A (PP2A) in complex with the B55&#946; targeting subunit. Importantly, our findings have been successfully replicated in human 293T cells, including the metabolic activation of CaMKII, and also the suppression of this activation by B55&#946; knockdown.</p><p>Our discovery represents a novel locus of CaMKII regulation and also provides a mechanism contributing to metabolic control of apoptosis. These findings may have implications for metabolic control of the many CaMKII-controlled and PP2A-regulated physiological processes, as both enzymes appear to be responsive to alterations in glucose metabolized via the PPP. Finally, our study reveals B55&#946; as a potential target for cancer therapy, because of its importance in suppressing metabolic suppression of caspase-2 activation and apoptosis.</p> / Dissertation

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