Dissertations / Theses on the topic 'Enzymes; Pentose phosphate pathway'

High aerobic glycolytic capacity is correlated with poor prognosis and increased tumour aggressiveness. 6Phosphofructo-1-kinase catalyses the first irreversible step of glycolysis, and is activated by fructose-2,6-bisphosphate, a product of the kinase activity of four bifunctional isoenzymes, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFK-2/FBPase-2:PFKFB1-4). These are potential anti-tumour targets, but their individual and collective role requires further investigation. This thesis had three aims; to validate the PFK-2/FBPase-2 isoenzymes as anti-cancer targets, to investigate the requirement for isoenzyme-specific targeting, and to initiate assay development, enabling future identification of novel inhibitors. A panel of cancer cell lines was examined and PFKFB3 and PFKFB4 were confirmed to be the most strongly induced isoenzymes in hypoxia, regulated by HIF-1α. Basal and hypoxic relative PFKFB3/PFKFB4 expression varied markedly, and three cell lines with varying expression ratios (MCF-7, U87, PC3) were selected for further study. siRNA knockdown of each isoenzyme individually, markedly reduced 2D and 3D cell growth. The effect of PFKFB3 knockdown was consistently more pronounced, particularly in hypoxia. Double PFKFB3/PFKFB4 knockdown was significantly less effective than PFKFB3 knockdown alone. Direct antagonism of PFKFB3 and PFKFB4 on F-2,6-BP concentration was observed, with PFKFB3 exhibiting high kinase activity, as anticipated, and PFKFB4 exhibiting high bisphosphatase activity. The degree of antagonism was dependent on the relative PFKFB3/PFKFB4 expression ratio. Extensive efforts were made to examine the wider metabolic effect of PFKFB3/PFKFB4 on flux towards glycolysis or the pentose phosphate pathway (PPP), including using metabolite, lipid droplet, ¹³C NMR and mass spectrometry assays. No significant change in metabolic flux was detected, the evidence presented therefore suggesting the impact of the antagonistic effects of the isoenzymes on [F-2,6-BP] extends beyond regulation of metabolic flux alone. This study concluded that the most effective therapeutic strategy will be one that involves a PFKFB3-specific inhibitor, preferably hypoxia-targeted. Accordingly, steps were taken to validate and optimise a robust medium-throughput assay system.

The role of the pentose phosphate pathway as a source of NADPH required for cytoplasmic processes such as lipogenesis and detoxification reactions has been examined. G6PDH and 6PGDH are known to be strongly inhibited by the high NADPH/NADP ratio which is thought to occur in the cytoplasm but no effector at physiological concentrations has yet been found which can overcome this inhibition. Initially a possible role for F2, 6P2 as an activator of G6PDH, 6PGDH and FAS was investigated but no significant effect of this regulatory metabolite on any of these enzymes was discovered. An attempt was also made to demonstrate the reported reversal of the inhibition by GSSG and the cofactor reported by Eggleston and Krebs (1974). This too could not be demonstrated. In the course of the work, a cytosolic NADPH-consuming reaction has been characterized. This has been shown to involve the reaction of a peptide-substrate with a cytoplasmic reductase specific for NADPH and a high affinity for the peptide. The physiological role of this reaction remains to be established, but it has been observed that the reaction exhibits a diurnal variation, the pattern of which is the reverse of that observed with lipogenesis. The low molecular weight peptide, which appears to be distinct from glutathione, contains cystine residues which are apparently reduced in the presence of NADPH, resulting in the appearance of free thiol groups. The peptide may be phosphorylated but the nature of the linkage between the peptide and phosphate has not been established. A possible role for this and other NADPH-dependent reactions in the regulation of the pentose phosphate pathway is discussed in this thesis.

In this thesis, tumor metabolic reprogramming has been exploited in order to propose new targets in cancer treatment. On one hand, we explored the pentose phosphate pathway (PPP) enzymes as putative therapeutic targets against breast and colon cancer. We observed that inhibition of ox-PPP enzymes G6PD in colon cancer cells and 6PGD in breast cancer cells halted cell prolifertion, resulted in cell cycle arrest and apoptosis. We also demosntrated that in colon cancer cells G6PD is strongly regulated by the glutamine availability mediated by NRF2 transcription factor. Moreover, 6PGD inhibition decreased mammosphere formation capacity of breast cancer cells implying that stem cell characteristics of breast cancer cells were altered by 6PGD inhibition. Besides that, 6PGD inhibition also altered the central carbon metabolism of breast cancer cells leading to decreased glucose consumption and increased glutamine consumption. Observing that both pathways are deeply related to glutamine metabolism, we decided to investigate the metabolic network adaptations that breast cancer cells undergo when the glutamine is scarce. Knowing that hypoxic conditions are common features of tumor microenvironments, we also investigated the characterization of a hypoxia mimicking condition which leads to defective mitochondria. In fact, in these two conditions, we produced huge amount of transcriptomics, metabolomics and fluxomics data in order to produce a genome scale metabolic model (GSMM) combining multi-omics data in the frame of a European project which helps us to understand the regulation of metabolic alterations in breast cancer cells. While produced data is to be used in production of GSMM, we also took advantage of the data to study the metabolic adaptations that breast cancer cells undergo in the deprivation of glutamine or when mitochondria are defected. We propose that increased pyruvate cycle with glutamine deprivation and increased reductive carboxylation with not fully functional mitochondria could be targeted in combination therapies to fight against cancer. All in all, besides showing the importance of metabolism in cancer cell proliferation and survival, the results presented in this study also highlights the importance of Systems Biology approaches to understand the molecular mechanisms underlying complex multifactorial diseases in order to develop new potential therapeutic targets.

Cancer est un terme qui rassemble plusieurs ensembles hétérogène de maladies et il est caractérisé par la perte de contrôle physiologique et la transformation maligne des cellules saines. Il est essentiel de comprendre le cancer de la biologie cellulaire afin d'identifier de nouveaux biomarqueurs pour le diagnostic précoce et la conception de nouvelles stratégies thérapeutiques. Reprogrammation métabolique est une caractéristique émergente de cancer, ce qui signifie que les cellules cancéreuses passent leur métabolisme de base pour répondre aux exigences accrues de la croissance et la division cellulaire. Par conséquent, explorer reprogrammant métabolique que les cellules cancéreuses subissent est une stratégie clé pour identifier de nouvelles cibles pour le traitement du cancer. Dans cette thèse, de nouvelles possibilités pour le traitement du cancer ont été explorés en analysant la reprogrammation métabolique de la tumeur. À cet égard, nous avons étudié et proposé voie des pentoses phosphates (PPP) enzymes cibles thérapeutiques putatifs contre les cancers du sein et du côlon. En outre, nous avons exploré le métabolisme de la glutamine dans les cellules du cancer du sein et les adaptations du réseau métaboliques qu'ils subissent dans le but de contourner la privation de glutamine et la déficience mitochondriale générale. Ainsi, le ciblage PPP est l'intérêt des chercheurs d'utiliser à la fois oxydantes et non oxydantes phases de cette voie métabolique comme une cible de médicament thérapeutique. Pour tester cela, nous inhibés bœuf PPP enzymes 6PGD dans les cellules cancéreuses du sein et G6PD dans les cellules du côlon.Nous avons effectué la caractérisation de la reprogrammation métabolique induite par l'inhibition de l'enzyme de bœuf PPP par l'ARN interferase (ARNi) silençage médiation, afin d'explorer le potentiel de cette enzyme comme une cible de médicament thérapeutique dans deux lignées de cellules de cancer du sein. Nous avons demontré que l'inhibition 6PGD a entraîné une diminution taux de prolifération, arrêt du cycle cellulaire et induction de l'apoptose médiée par l'activation de p53, en diminuant les capacités de formation mammosphere et le métabolisme altéré de carbone central par modulation de Warburg phenomenan et en améliorant le métabolisme de la glutamine. D'autre part, nous avons montré l'effet de l'inhibition de la G6PD sur la prolifération des cellules du cancer du côlon et du PPP est régulée par la disponibilité de la glutamine dans les cellules cancéreuses du côlon.De plus, nous avons caractérisé les adaptations métaboliques que les cellules cancéreuses du sein subissent la privation de glutamine ou lorsque les mitochondries sont fait défection. Nous avons effectué une analyse des flux métaboliques utilisant métabolomique et Fluxomique et nous avons utilisé la biologie des systèmes afin d'estimer une vision globale des modifications de flux dans différentes conditions de culture. Nous avons observé une augmentation du cycle de pyruvate avec privation glutamine, ce qui indique que le ciblage des enzymes de cette voie telle que l'enzyme malique pourrait être une approche prometteuse combinée à l'inhibition de l'enzyme de glutaminase. D'autre part, nous avons observé que mimant une hypoxie par des cellules de cancer du sein de traitement redirigée oligomycine pour augmenter la carboxylation réductrice. Considérant que l'hypoxie est une condition commune dans l'environnement de la tumeur, le ciblage mécanisme de carboxylation réductrice pourrait être une nouvelle stratégie de lutte contre le cancer. Collectivement, les résultats présentés dans cette thèse démontre l'importance du métabolisme de la prolifération des cellules cancéreuses et la survie. Ce travail met également en évidence l'importance de la biologie des systèmes se rapproche de comprendre les mécanismes moléculaires sous-jacents des maladies multifactorielles complexes afin de souligner de nouvelles cibles thérapeutiques potentielles
Moreover, we characterized the metabolic adaptations that breast cancer cells undergo in the deprivation of glutamine or when mitochondria are defected. We conducted metabolic flux analysis using metabolomics and fluxomics approaches and we employed Systems Biology approaches in order to estimate a global view of flux alterations in different culture conditions. We observed an increased pyruvate cycle with glutamine deprivation, thus indicating that targeting the enzymes of this pathway such as malic enzyme could be a promising approach combined with inhibition of glutaminase enzyme. On the other hand, we observed that mimicking hypoxia by oligomycin treatment redirected breast cancer cells to increase reductive carboxylation. Considering that hypoxia is a common condition in the tumor environment, targeting reductive carboxylation mechanism could be a novel strategy to fight against cancer. Collectively, all the results provided in this thesis demosntrate the importance of metabolism in cancer cell proliferation and survival. This work also highlights the importance of Systems Biology approaches to comprehend the molecular mechanisms underlying complex multifactorial diseases in order to point out new potential therapeutic targets

G-quadruplex stabilising ligands are of interest as potential anti-cancer drugs. Gquadruplexes are folded DNA structures which can form in guanine-rich regions of DNA or RNA, and stabilisation of these structures at telomeres can result in the inhibition of telomerase activity. In this thesis, Saccharomyces cerevisiae was used as a model to examine the in vivo cellular response to treatment with the G-quadruplex stabilising ligand TMPyP4. The findings indicate that the pentose phosphate pathway (PPP) is key for resistance to TMPyP4, since the absence of PPP genes resulted in increased sensitivity to treatment with the ligand. However, the TMPyP4-sensitivity exhibited by pppΔ strains is most likely due to oxidative stress caused by the photosensitivity of the porphyrin. There are also potential links between PPP activity, the response to uncapped telomeres and the DNA damage response (DDR). The cdc13- 1 mutant strain, in which the telomere binding protein Cdc13 is defective, was used to explore these connections. Here, I demonstrate that deletion of key PPP genes results in suppression of the temperature-sensitive growth phenotype of cdc13-1. In addition, the activity of the enzyme which catalyses the initial step of the oxidative phase of the PPP, Zwf1, increases in cdc13-1 strains. Studies of Zwf1 activity in strains arrested in late anaphase and G1, however, suggest that the increase in Zwf1 activity is due to the phase of the cell cycle in which the strains are arrested, rather than the DDR. The work described here demonstrates that the PPP is intrinsically linked to the response to a variety of cellular stresses. Due to its role in the Warburg effect, a metabolic shift observed in tumour cells, examination of PPP function is important not only for the study of normal tissues but also immortal cancer cells, and lessons in budding yeast can lead to important insights.

The present doctoral thesis is focused on the metabolic adaptations induced by oncogene activation as well as the potential role of the metabolic network as antitumor therapy. Over the last years, it has emerged a renewed interest in the field of metabolism, particularly in cancer metabolism. Great efforts have been focused on the association of mutated oncogenes or tumor suppressor genes and tumor metabolic profiles, in the search of metabolic dependencies that offer new potential avenues for cancer treatment. The pursuit of discovering tumor metabolic alterations in which cancer cells rely on has represented the cornerstone of this interesting discipline. Thus, this thesis is part of this recent and promising scientific current and is intended to shed light on the metabolic alterations accompanying oncogene mutation and on potential metabolic pathways that might be of therapeutic interest in the future. Hence, the objectives of this thesis can be divided into two specific aims: i) analysis of the metabolic reprogramming of RAS oncogenic activation using stable transfected cell lines with mutated copies of K-RAS and H-RAS and ii) validation of the pentose phosphate pathway as a potential therapeutic target and exploration of its role within tumor metabolism in colon and breast cancer cell models. Thus, according to the proposed objectives, the main conclusions obtained are as follow: 1. The study of flux distribution in combination with metabolic control analysis performed by analyzing solely the sign of fixed-sign control coefficients, is a reliable approach to identify the key enzymes involved in metabolic reprogramming. The use of this methodology has allowed us to identify an increase in glycolysis and PPP fluxes as metabolic features of KRAS-induced metabolic reprogramming and to propose G6PD, PK and LDH as the key enzymes responsible for this metabolic transition. 2. H-RAS oncogenic activation reprograms glucose and glutamine metabolism by enhancing glycolytic and PPP fluxes as well as mitochondrial metabolism. Glutamine is responsible for sustaining the activated mitochondrial metabolism in BJ-HRasV12, while glucose-derived carbons in the mitochondria are primarily used to fuel lipogenesis. Moreover, lipogenesis is overactivated in BJ-HRasV12 cells, which are more sensitive to FAS inhibition than BJ cells. 3. G6PD enzyme is overactivated in colon cancer cells with oncogenic activation of the RAS signaling pathway. Nevertheless, G6PD seems to be dispensable for proliferation and survival in BRAF-mutated HT29 cell line. Furthermore, a new connection between PPP and glutamine metabolism has been unveiled, as G6PD is overexpressed in HT29 cells under glutamine-deprived conditions by a mechanism involving a concomitantly increase in ROS levels and NRF2 induction. 4. G6PD enzyme is important in proliferation, survival and regulation of ROS levels in breast cancer MCF7 cells. However, it exerts a low regulation over ribose synthesis flux through the oxidative branch of PPP. G6PD inhibition enhances glycolytic flux, promotes lactate secretion and increases glutamine consumption, which is used to maintain energy homeostasis, although it is not essential for cell proliferation. 5. TKT enzyme is dispensable for proliferation of breast cancer MCF7 cells, but it exerts a high control over ribose synthesis flux through the nonoxidative branch of PPP. TKT impairment reduces glycolytic flux and increases the consumption of glutamine, which is intended to maintain energy homeostasis but it is not essential for cell proliferation.
La presente tesis doctoral se centra en las adaptaciones metabólicas inducidas por la activación de oncogenes así como en el potencial del entramado metabólico como diana antitumoral. A lo largo de los últimos años, ha resurgido un renovado interés en el estudio del metabolismo, particularmente en el metabolismo de las células tumorales, dando lugar a una nueva disciplina conocida como metabolismo tumoral. Numerosas investigaciones se han centrado en la asociación entre mutaciones en oncogenes o genes supresores de tumores con perfiles metabólicos característicos, en busca de dependencias metabólicas que ofrezcan nuevas posibilidades para el tratamiento de los tumores. La búsqueda de alteraciones metabólicas que constituyan vulnerabilidades de la célula tumoral representa la piedra angular de esta interesante disciplina. Así, esta tesis doctoral tiene como objetivo general elucidar las alteraciones metabólicas que acompañan a la mutación de oncogenes y explorar el potencial del entramado metabólico como diana antitumoral. Por tanto, los objetivos principales de este trabajo son los siguientes: i) análisis de la reprogramación metabólica inducida por la activación oncogénica de RAS empleando líneas celulares transfectadas de manera estable con copias mutadas de los oncogenes K-RAS y H-RAS y, ii) validación de la vía de las pentosas fosfato como potencial diana antitumoral y estudio de su papel en el metabolismo tumoral de modelos celulares de cáncer de colon y de mama. Así, en este trabajo de tesis doctoral hemos concluido que la activación oncogénica de RAS promueve una profunda reprogramación del metabolismo induciendo cambios significativos en la glucólisis, la vía de las pentosas fosfato, el metabolismo de la glutamina y la lipogénesis. Por otro lado, hemos determinado que la inhibición de la vía de las pentosas fosfato tiene distintos efectos según el tipo de tumor. La inhibición de la G6PD en la línea celular de cáncer colon HT29 no produjo efectos sobre la proliferación mientras que su inhibición en células de cáncer de mama MCF7 indujo una notable reducción de la proliferación y un incremento de la muerte celular. Por otra parte, en la inhibición en MCF7 del otro enzima clave de la vía de las pentosas fosfato, la TKT, no se observaron cambios significativos en términos de proliferación y viabilidad celular. Además, en este trabajo también se ha puesto de manifiesto una conexión funcional entre la vía de las pentosas fosfato y el metabolismo de la glutamina en ambos modelos celulares, sugiriendo un papel complementario de estas dos vías metabólicas.

Hydrogen production from abundant renewable biomass would decrease reliance on crude oils, achieve nearly zero net greenhouse gas emissions, create more jobs, and enhance national energy security. Cell-free synthetic pathway biotransformation (SyPaB) is the implementation of complicated chemical reaction by the in vitro assembly of numerous enzymes and coenzymes that microbes cannot do. One of the largest challenges is the high cost and instability of enzymes and cofactors. To overcome this obstacle, strong motivations have driven intensive efforts in discovering, engineering, and producing thermostable enzymes. In this project, ribose-5-phosphate isomerase (RpiB), one of the most important enzymes in the pentose phosphate pathway, was cloned from a thermophile Thermotoga maritima, and heterologously expressed in Escherichia coli, purified and characterized. High-purity RpiB was obtained by heat pretreatment through its optimization in buffer choice, buffer pH, as well as temperature and duration of pretreatment. This enzyme had the maximum activity at 80°C and pH 6.5-8.0. It had a half lifetime of 71 h at 60°C, resulting in its turn-over number of more than 2 x108 mol of product per mol of enzyme. Another two thermostable enzymes glucose-6-phosphate dehydrogenase (G6PDH) and diaphorase (DI) and their fusion proteins G6PDH-DI and DI-G6PDH were cloned from Geobacillus stearothermophilus, heterologouely expressed in E. coli and purified through its His-tag. The individual proteins G6PDH and DI have good thermostability and reactivity. However, the presence of DI in fusion proteins drastically decreased G6DPH activity. However, a mixture of G6PDH and a fusion protein G6PDH-DI not only restored G6PDH activity through the formation of heteromultimeric network but also facilitated substrate channeling between DI and G6PDH, especially at low enzyme concentrations. My researches would provide important building blocks for the on-going projects: high-yield hydrogen production through cell-free enzymatic pathways and electrical energy production through enzymatic fuel cells.
Master of Science

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The association between proline metabolism in the plant and the pentose phosphate pathway has been proposed as a model to stimulate one of the biosynthetic routes of secondary metabolism related to the production of the different phenolic compounds in plants, known as the shikimic acid pathway. In transgenic plants in which overexpression of the P5CS gene Δ1-pyrroline-5-carboxylate synthetase encoding the key enzyme of proline biosynthesis occurs, in addition to increased tolerance to abiotic stresses, there is the possibility of promoting the synthesis of phenolic compounds as a pleiotropic effect. The objective of this work was to evaluate the role of proline "per se" in relation to the production of phenolic compounds in transgenic tobacco plants accumulating this amino acid (Nicotiana tabacum cv. Petit Havana SR1) and to verify if water stress would modify this answer. The experiment was carried out under greenhouse conditions, at Campus II of Universidade do Oeste Paulista, Presidente Prudente - SP. A completely randomized design was used in the factorial scheme 3x2 consisting of 3 genotypes (two transgenic events with constitutive expression 35S::P5CSF129A and untransformed control plants) and two levels of water regime, with daily water replenishment of 100% and 30% of field capacity (water stress). The role of proline "per se" was evaluated by means of biometric growth analyzes (plant height, shoot and root dry mass and leaf number) in addition to proline analyzes, glucose-6-phosphate dehydrogenase activity (G6PDH), phenylalanine ammonia lyase (PAL), total phenolic compounds in leaves and lignin. In a natural response to water stress, a reduction in biometric parameters was observed for all genotypes. The accumulation of proline occurred in a greater proportion in the transgenic plants as expected by the constitutive expression of the P5CS gene, but the activity of the G6PDH enzyme was lower in the transgenic plants. The link between increased proline endogenous content and increased phenol synthesis occurred both under normal hydration conditions (for E2 event) and in the presence of water stress (for both transgenic events), as well as PAL activity. Lignin contents increased in all genotypes in response to stress. Thus, the results of this research lead us to affirm the existence of distinct responses between exogenous application of proline reported in the literature and endogenous cellular metabolism. It was not possible to confirm the hypothesis that the proline metabolism linked to the pentose phosphate pathway induces the synthesis of phenolic compounds, since the activity of the G6PDH enzyme was lower in the transgenic plants in the two water conditions. It is suggested that the precursors to the pathway of phenolic compounds linked to proline may be provided by other metabolic pathways such as glycolysis and the Calvin cycle. Further study will be needed to clarify this issue.
A associação entre o metabolismo de prolina na planta e a via pentose fosfato tem sido proposta como um modelo para se estimular uma das rotas do metabolismo secundário relacionada à produção dos diferentes compostos fenólicos em plantas, denominada via do ácido chiquímico. Em plantas transgênicas nas quais ocorrem a superexpressão do gene P5CS Δ1-pirrolina-5-carboxilato sintetase, que codifica a enzima-chave da biossíntese de prolina, além do aumento de tolerância a estresses abióticos, existe a possibilidade de promoção da síntese de compostos fenólicos como um efeito pleiotrópico. O objetivo deste trabalho foi avaliar o papel da prolina “per se” em relação à produção de compostos fenólicos em plantas transgênicas de tabaco acumuladoras deste aminoácido (Nicotiana tabacum cv. Petit Havana SR1) e verificar se o estresse hídrico modificaria essa resposta. O experimento foi realizado em condições de casa de vegetação, no Campus II da Universidade do Oeste Paulista, Presidente Prudente - SP. Utilizou-se o delineamento experimental em arranjo inteiramente casualizado, no esquema fatorial 3x2, formado por 3 genótipos (dois eventos transgênicos com expressão constitutiva 35S::P5CSF129A e plantas controle não transformadas) e dois níveis de regime hídrico, com reposições diárias de água de 100% e de 30% da capacidade de campo (estresse hídrico). O papel da prolina “per se” foi avaliado por meio de análises biométricas de crescimento (altura de plantas, massas seca de parte aérea e raiz e número de folhas) além das análises de prolina, atividade das enzimas glicose-6-fosfato-desidrogenase (G6PDH), fenilalanina amônia liase (FAL), compostos fenólicos totais nas folhas e lignina. Em resposta natural ao estresse hídrico, foi observado uma redução nos parâmetros biométricos para todos os genótipos. O acúmulo de prolina ocorreu em maior proporção nas plantas transgênicas como esperado pela expressão constitutiva do gene P5CS, porém a atividade da enzima G6PDH foi menor nas plantas transgênicas. A vinculação entre um maior conteúdo endógeno de prolina com a síntese aumentada de fenóis ocorreu tanto em condições normais de hidratação (para o evento E2) como na presença de estresse hídrico (para ambos os eventos transgênicos), assim como a atividade da FAL. Os teores de lignina aumentou em todos os genótipos em resposta ao estresse. Desta forma, os resultados dessa pesquisa levam-nos a afirmar a existência de respostas distintas entre aplicação exógena de prolina relatada na literatura e o metabolismo endógeno celular. Não foi possível confirmar a hipótese de que o metabolismo de prolina vinculado à via pentose fosfato induz a síntese de compostos fenólicos, pois a atividade da enzima G6PDH foi menor nas plantas transgênicas nas duas condições hídricas. Sugere-se que os precursores para a via dos compostos fenólicos ligados a prolina podem ser fornecidos por outras vias metabólicas tais como a glicólise e o ciclo de Calvin. Estudos mais aprofundados serão necessários para esclarecer esta questão.

Circadian rhythms are self-sustained endogenous biological oscillations with a period of approximately 24 hours. These rhythms are observed widely across kingdoms and at all levels of biological scale. Recent work has shown there to be circadian variation in metabolism, both at the organismal and cellular level. It has also been posited that rhythmic production of metabolites might be essential for maintenance of circadian rhythmicity within cells, even in the absence of nascent transcription. The first portion of this thesis investigates the contribution of primary carbohydrate metabolism to cellular timekeeping, with particular emphasis on the pentose phosphate pathway. I also describe and validate a new 13C labelling technique for accurate determination of the relative flux through early primary metabolic pathways. This is accompanied by the development and optimisation of a microfluidic system for long-term perfused tissue culture, which allows for longitudinal study of metabolic flux within the same population of cells with simultaneous recording of clock gene activity. This perfused system provides several advantages over static tissue culture. The second portion considers the effects of the metabolic hormone insulin on circadian rhythmicity, both at the level of the cell and of the whole organism. It shows that administration of insulin is sufficient to shift the phase of circadian gene expression and elicits induction of clock protein PER2. Strikingly, manipulation of insulin signalling is sufficient to determine all the essential parameters of the cellular clock (phase, period and amplitude) in a dose-dependent but glucose independent fashion. Using pharmacological and genetic approaches, a molecular explanation for this effect is determined. This data suggests that insulin is a primary determinant of rhythms in peripheral tissues and is most likely a major signal for circadian entrainment to feeding in mammals, for which I now propose a mechanistic basis.

Allosteric regulation of important enzymes is a mechanism frequently employed by organisms to exert control over their metabolism. The shikimate pathway is ultimately responsible for the biosynthesis of the aromatic amino acids in plants, microorganisms and apicomplexans. Two enzymes of the pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) and chorismate mutase (CM) are located at critical positions along the aromatic amino acid biosynthetic pathway and are often tightly feedback regulated in order to control the flux of metabolites through the pathway. This research presents studies on the allosteric function of these two enzymes. These studies emphasise the complexity of the intersecting network of allosteric response, which alters the catalytic activity of each enzyme in response to metabolic demand for the aromatic amino acids.

Les Leucémies Aigües Myéloïdes (LAM) sont des hémopathies malignes hétérogènes de mauvais pronostic qui se caractérisent par une expansion clonale de progéniteurs immatures. De nombreuses dérégulations de voies de signalisation sont retrouvées dans les cellules leucémiques et leur confèrent un avantage de prolifération et de survie. La voie de signalisation mTORC1, qui contrôle la traduction protéique, l’autophagie et plusieurs voies métaboliques, est ainsi constitutivement activée dans les cellules leucémiques. La reprogrammation métabolique notamment via « l’effet Warburg » est un phénomène bien décrit dans les cellules cancéreuses. L’augmentation de l’utilisation de la glycolyse, confère aux cellules tumorales un avantage de survie en favorisant une production rapide d’ATP et d’intermédiaires métaboliques nécessaires pour les biosynthèses de nucléotides, d’acides-aminés et de lipides. C’est donc dans ce contexte que j’ai étudié le métabolisme du glucose dans les cellules de LAM et l’implication de la voie de signalisation mTORC1 dans la dérégulation de ce métabolisme. J’ai tout d’abord identifié par une étude transcriptomique dans la lignée leucémique MOLM-14 que la signalisation mTORC1 contrôle plusieurs voies métaboliques notamment celles permettant l’utilisation du glucose. Ceci a été vérifié dans plusieurs lignées de LAM puisque l’inhibition ou la sur-activation de mTORC1 entrainent respectivement une diminution ou une augmentation de la consommation de glucose et de la production de lactate. De façon intéressante, le niveau d’activation de la voie mTORC1 détermine la sensibilité des cellules leucémiques à l’inhibition de la glycolyse. En effet, lorsque mTORC1 est activé, le blocage de la glycolyse induit de l’autophagie et l’apoptose des cellules leucémiques. A l’inverse, le blocage de mTORC1 induit une reprogrammation métabolique des cellules leucémiques qui utilisent alors principalement la phosphorylation oxydative pour produire l’ATP dont elles ont besoin. Leur survie devient alors indépendante du glucose. A l’inverse des cellules primaires de LAM, les cellules hématopoïétiques immatures normales CD34+ sont moins sensibles au blocage de la glycolyse. Le ciblage du métabolisme du glucose pourrait donc constituer une stratégie thérapeutique intéressante dans les LAM. Je me suis ensuite intéressée aux effets anti-leucémiques induits par l’inhibition de la voie des pentoses phosphates (PP) et plus particulièrement au ciblage de la G6PD (glucose-6-phosphate déshydrogénase) par le composé le 6-aminonicotinamide (6-AN). En effet, une étude de flux métabolique a permis de mettre en évidence qu’une proportion importante de glucose est dirigé vers la voie des PP, laissant suggérer que l’addiction des cellules leucémiques au glucose pourrait être liée à une utilisation augmentée de cette voie annexe. J’ai alors observé que le 6-AN induit une cytotoxicité in-vitro y compris dans les cellules primaires de patients, sans avoir d’effets sur les cellules hématopoïétiques normales et in-vivo dans un modèle de xénogreffe de la lignée MOLM-14 chez la souris NUDE. Cette étude a donc permis de montrer que l’activation constitutive de mTORC1 rend la survie des cellules de LAM dépendante de la glycolyse et crée une sensibilité spécifique à l’inhibition de la G6PD. La dérégulation de la signalisation mTORC1 étant quasi-constante dans les LAM, cibler la G6PD pourrait donc représenter une stratégie thérapeutique intéressante
Acute Myeloid Leukemia (AML) are heterogeneous hematological diseases with poor prognosis characterized by a clonal expansion of immature progenitors. Many deregulation of signaling pathways are found in leukemic cells and give them an advantage of proliferation and survival. The MTORC1 signaling pathway, which controls protein translation, autophagy and several metabolic pathways, is constitutively activated in leukemic cells. Metabolic reprogramming in particular the "Warburg effect" is a phenomenon well described in cancer cells. High rate of glycolysis has been considered to give tumour cells advantages through rapid production of ATP and intermediates for the synthesis of nucleotides, amino acids, and lipids. In this context, I studied glucose metabolism in AML cells and the involvement of the mTORC1 signaling pathway in the deregulation of this metabolism. First, I identified by a transcriptomic analysis in the MOLM-14 cell line that mTORC1 signaling controls several metabolic pathways including those for glucose utilization. This has been verified in several AML cell lines, since inhibition or over-activation of mTORC1 respectively induces a decrease or an increase in glucose consumption and lactate production. Interestingly, the level of activation of the mTORC1 signaling pathway determines the sensitivity of AML cells to the inhibition of glycolysis. Indeed, when mTORC1 is activated, the blockade of glycolysis induces autophagy and apoptosis of leukemic cells. Conversely, blocking mTORC1 induces metabolic reprogramming of leukemic cells, which then mainly use oxidative phosphorylation to produce ATP for their needs. AML cell survival become independent of glucose. Unlike primary AML cells, survival of normal immature hematopoietic cells CD34+ is only barely affected by the blockade of glycolysis. Thus, targeting the glucose metabolism may constitute an attractive therapeutic strategy in AML. I then investigated the anti-leukemic activity induced by the inhibition of the pentose phosphate pathway (PPP) and more particularly by the specific blockade of G6PD (glucose 6-phosphate dehydrogenase) with the 6-aminonicotinamide (6- AN) compound. Indeed, a metabolic flux analysis demonstrated that a significant proportion of glucose was directed towards the PPP. This result suggested that the addiction of leukemic cells toward glucose might be related to an increased use of PPP. I then observed that the 6-AN induced in vitro cytotoxicity including in primary AML cells from patients without effect on normal immature hematopoietic cells CD34+ and in vivo in a xenograft model of MOLM-14 cell line in the NUDE mouse. This study therefore demonstrated that the constitutive activation of mTORC1 makes AML cells survival dependent on glycolysis, and creates a specific vulnerability to the inhibition of G6PD. Given that deregulation of the mTORC1 signaling pathway is almost constant in AML, targeting G6PD may therefore represent an interesting therapeutic strategy

Le processus de germination de l'embryon de pommier se decompose en 2 phases essentielles, appelees germination sensustricto et croissance, qui different par leur sensibilite aux facteurs du milieu et aux inhibiteurs respiratoires. Le debut de la germination est caracterise par un etat cellulaire defavorable au fonctionnement de la voie des pentoses phosphates; le demarrage de la croissance coincide avec une intensification de cette voie metabolique et une augmentation de sa participation relative au catabolisme glucidique. L'achevement de la phase de germination sensu stricto pourrait dependre de cette orientation preferentielle du catabolisme glucidique vers la voie des pentoses phosphates

L'élucidation des propriétés du réseau métabolique est fondamentale pour la compréhension du fonctionnement cellulaire et pour l'élaboration de stratégies d'ingénierie métabolique. L'objectif de cette thèse était de mieux comprendre la régulation du métabolisme du NADPH, un métabolite "hub" qui joue un rôle central dans de nombreux processus cellulaires, chez Saccharomyces cerevisiae en fermentation. Nous avons utilisé une démarche systématique couplant modélisation et approches multi-“omics” pour étudier de façon quantitative la réponse à une perturbation de la demande en NADPH. Un système expérimental original, basé sur l'expression d'une butanediol déshydrogénase modifiée NADPH-dépendante a été utilisé pour augmenter de façon contrôlée la demande en NADPH. L'utilisation de ce dispositif, le développement et l'utilisation d'un modèle stœchiométrique de la levure dédié à la fermentation ont permis de prédire la répartition des flux pour différents niveaux de perturbation. Ces analyses ont montré, en premier lieu, la très grande capacité de la levure à faire face à des demandes très importantes de NADPH représentant jusqu'à 40 fois la demande anabolique. Pour des demandes modérées (allant jusqu'à 20 fois la demande anabolique), la perturbation est principalement compensée par une augmentation du flux à travers la voie des pentoses phosphate (VPP) et à moindre titre à travers la voie acétate (Ald6p). Pour une forte demande en NADPH, correspondant à 40 fois la demande anabolique, le modèle prédit la saturation de la VPP ainsi que la mise en place du cycle glycérol-DHA, qui permet l'échange du NADH en NADPH. Des analyses fluxomique (13C), métabolomique et transcriptomique, ont permis de valider ces hypothèses et de les compléter. Nous avons mis en évidence différents niveaux de régulation selon l'intensité de la perturbation : pour les demandes modérées, les flux sont réajustés par un contrôle au niveau enzymatique ; pour de fortes demandes, un contrôle transcriptionnel de plusieurs gènes de la VPP ainsi que de certains gènes des voies de biosynthèse des acides aminés est observé, cet effet résultant probablement de la moindre disponibilité en NADPH. Dans l'ensemble, ce travail a apporté un nouvel éclairage sur les mécanismes impliqués dans l'homéostasie du NADPH et plus généralement dans l'équilibre redox intracellulaire
The elucidation of the properties of metabolic network is essential to increase our understanding of cellular function and to design metabolic engineering strategies. The objective of this thesis was to better understand the regulation of the metabolism of NADPH, a “hub” metabolite which plays a central role in many cellular processes in Saccharomyces cerevisiae during fermentation. We used a systematic approach combining modeling and multi-“omics” analyses to study quantitatively the response to a perturbation of the NADPH demand. An original experimental system, based on the expression of a modified NADPH-dependent butanediol dehydrogenase was used to increase the demand for NADPH in a controlled manner. Through the use of this device and the development and use of a stoichiometric model of yeast dedicated to the fermentation, we predicted the flux distribution for different levels of perturbation. These experiments showed, first, the overwhelming ability of yeast to cope with very high NADPH demand, up to 40 times the anabolic demand. For a moderate level (up to 20 times the anabolic demand), the perturbation is mainly compensated by increased flux through the pentose phosphate pathway (PPP) and to a lesser extent through the acetate pathway (Ald6p). For a high NADPH demand, corresponding to 40 times the anabolic demand, the model predicts the saturation of the PPP as well as the operation of the glycerol-DHA cycle, which allows the exchange of NADH to NADPH. Fluxomics (13C), metabolomics and transcriptomics data were used to validate and to complement these hypotheses. We showed different levels of control depending on the intensity of the perturbation: for moderate demands, flux remodeling is mainly achieved by enzymatic control; for a high demand, a transcriptional control is observed for several genes of the PPP as well as some genes of the amino acids biosynthetic pathways, this latter effect being likely due to the low NADPH availability. Overall, this work has shed new light on the mechanisms governing NADPH homeostasis and more generally the intracellular redox balance

Los isoprenoides son una de las mayores familias de compuestos descritos en la naturaleza. Estos compuestos están presentes en todos los organismos vivos y se sintetizan a partir de dos moléculas de 5 átomos de carbono: el isopentenil difosfato (IPP) y el dimetilalil difosfato (DMAPP). Actualmente se conoce que arqueobacterias, hongos y animales presentan la ruta del mevalonato de síntesis de estos precursores, mientras que eubacterias, algún protozoo (como el causante de la malaria) y protistas presentan la ruta del metileritritol 4-fosfato (MEP) de síntesis de IPP y DMAPP. Estas rutas coexisten separadas espacialmente en plantas, helechos y algunas algas. La ruta del MEP de biosíntesis de los precursores de isoprenoides se muestra como una atractiva diana para la búsqueda de nuevos compuestos antimaláricos, antibióticos y herbicidas debido a su presencia en los principales agentes patogénicos y su ausencia en animales, además del carácter esencial de los isoprenoides para la vida. En esta tesis se ha realizado la búsqueda asistida por ordenador de compuestos que puedan interferir en la formación del complejo homodimérico del cuarto paso enzimático de la ruta del MEP. La metodología utilizada es muy útil en la búsqueda de inhibidores específicos. Se han caracterizado la unión de diferentes compuestos obtenidos con la enzima. Además se ha caracterizado el estado de oligomerización de la enzima. Paralelamente también se ha caracterizado un homólogo del primer paso enzimático de la ruta del MEP de un organismo termofílico caracterizando sus principales parámetros cinéticos y residuos importantes para la actividad enzimática mediante mutagénesis dirigida. Como último punto, se ha caracterizado el proceso de proteólisis de diferentes homólogos de este primer paso enzimático de la ruta del MEP asociándolo a modificaciones postraduccionales intramoleculares de las mismas proteínas, abriendo la posibilidad de un proceso de regulación posttraduccional de la actividad enzimática en este tipo de enzimas.

Molecular aspects of nitrogen metabolism in vertebrates is an interesting area of physiology and evolution to explore due to the different ways in which animals excrete nitrogenous waste as they transition from an aquatic to a terrestrial lifestyle. Two main products of nitrogen metabolism in fishes are ammonia and urea. Ammonia is produced during protein catabolism and build up of ammonia is toxic. Some aquatic vertebrates convert ammonia into a less toxic compound urea via de novo synthesis through the ornithine-urea cycle (O-UC). Five enzymes are involved in the O-UC: carbamoyl phosphate synthetase (CPS), ornithine carbamoyl transferase (OCT), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and arginase (ARG). An accessory enzyme, glutamine synthetase (GS) also participates in the "fish-type" O-UC. Teleosts excrete ammonia passively over their gills into the aquatic environment. The teleost, Opsanus beta, has been shown to increase urea production after 48 hours of crowding. This thesis explored how crowding stress affected nitrogen metabolite levels of ammonia and urea and O-UC gene expression and enzyme activity in O. beta. Lungfishes while in an aquatic environment avoid ammonia toxicity by releasing excess ammonia across their gills, but when stranded on land they produce urea through the O-UC. Urea production via the O-UC has a metabolic cost of at least four ATP molecules. This thesis explored the response of a lungfish, Protopterus annectens, to six days of aerial exposure and re-immersion conditions by measuring concentrations of O-UC mRNA expression and enzyme activity and nitrogen metabolites ammonia and urea. CPS acts as the entry point to the O-UC and based on enzymatic studies, most aquatic vertebrates utilize one isoform of this enzyme (CPSIII) while terrestrial vertebrates utilize a different isoform of this enzyme (CPSI). Lungfishes are a particularly interesting group of air-breathing fishes, not only because of their link to the origins of tetrapods, but also because CPS I may have originated within this group. Both CPS III and CPS I have been enzymatically described within this group. This thesis uses phylogenetics to investigate how CPS nucleotide sequences in lungfishes evolved compared to other vertebrates.

Nous avons étudié les effets du glycérol sur la croissance et le métabolisme des cellules végétales non chlorophylliennes, en utilisant des cellules isolées d'érable sycomore (Acer Pseudoplatanus, l. ) et d'Echinochloa. Le glycérol pénètre dans les cellules par diffusion. Il est ensuite phosphoryle en SN-glycérol-3-P qui alimente la respiration, via les réactions terminales de la glycolyse. La néoglucogenèse est impossible car le SN-glycérol-3-P est un inhibiteur compétitif de la glucose-6-phosphate isomérase. Ainsi, la synthèse de saccharose, d'amidon et de composes pariétaux s'arrête. De plus le cycle oxydatif des pentoses cesse d'être alimente, ce qui entraine un arrêt de la synthèse des pentoses et de la réduction des nitrates. Lorsque le glycérol est utilise comme seule source de carbone, la croissance des cellules est alors stoppée. Pourtant le seul fonctionnement de la respiration mitochondriale suffit à entretenir durant plusieurs semaines la survie des cellules, qui ne présentent aucun signe d'autophagie. Par contre, une carence de glycérol entrainera brutalement le processus autophagique. Par ailleurs, nous avons observe que le glycérol entraine l'accumulation d'oligopolysaccharides de types B-1,3 et de phosphohomoserine (un intermédiaire de la synthèse d'isoleucine, issu de l'aspartate) lorsque le milieu de culture contient du saccharose. En utilisant l'homosérine (dont nous avons caractérise le transport actif dans les cellules et la métabolisation) et les herbicides inhibant l'acétolactate synthase, nous avons montre que le site principal de régulation de la synthèse in vivo de l'isoleucine est situe au niveau de la thréonine déshydratase. Une observation a été mentionnée en annexe : l'induction de la respiration insensible au cyanure par les inhibiteurs de l'acétolactate synthase

Transaldolase-Mangel (TALDO) ist ein extrem seltener, angeborener Stoffwechseldefekt, von dem weltweit nur 34 Fälle bekannt sind. Der Defekt geht auf den Verlust des Enzyms Transaldolase 1 aus dem nicht-oxidativen Pentosephosphat-Weg (nicht-oxPPW) zurück und äußert sich in einem weiten Spektrum klinischer Symptome. Die schwerwiegendsten Folgen sind Leber- und Nierenmangelfunktionen, die zum sehr frühen Tod führen können. Desweiteren leiden 15 % der Patienten an wiederkehrenden Infektionen. Neutrophile Granulozyten (Neutrophile) sind die häufigsten weißen Blutkörperchen im Menschen und essentiell für die angeborene Immunantwort gegen Infektionserreger. Ich habe hier funktionale Aspekte von TALDO-Neutrophilen untersucht. Der oxidative Pentosephosphat-Weg (oxPPW) stellt das Reduktionsäquivalent NADPH bereit, welches indirekt für die Entstehung von reactive oxygen species (ROS)-abhängigen Neutrophil Extracellular Traps (NETs) verantwortlich ist. Der Beitrag des nicht-oxPPW zur ROS-abhängigen NET-Bildung ist bislang nicht bekannt. In dieser Arbeit konnte ich für Neutrophile aus drei TALDO-Patienten eine jeweils komplett abwesende Entstehung ROS-abhängiger NETs und einen deutlich verringerten oxidativen Burst nach PMA-Stimulation zeigen. Um diese Beobachtungen in einem unabhängigen Modelsystem zu bestätigen, habe ich mit Hilfe des CRISPR-Cas9-Systems, ‚knock-out‘ Mutanten von Transaldolase 1 und dessen Partnerenzym Transketolase in der Neutrophil-ähnlichen Zelllinie PLB-985 hergestellt. Die dergestalt genetisch manipulierten Zellen waren nicht mehr zu PMA-induziertem Zelltod in der Lage. Dies ist somit der erste genetische Beweis für die Abhängigkeit des oxidativen Burst und der Bildung von NETs vom nicht-oxPPW. Diese Erkenntnis trägt zum einen zum mechanistischen Verständnis der NET-Entstehung bei und liefert zum anderen eine potentielle Erklärung für einige der bei TALDO beobachteten Symptome. Desweiteren wurden einige der metabolischen Erfordernisse für die Bildung von NETs mit Hilfe von Inhibitoren untersucht. Die erhaltenen Erkenntnisse zeigen, dass das initiale Maximum des oxidativen Bursts für NET-Bildung unerheblich ist und vielmehr die ROS-Generierung nach ca. 50 Minuten entscheidende Bedeutung für diese hat.
Transdaldolase 1-deficiency (TALDO) is a rare genetic disease with only 34 described cases globally. Transaldolase 1 is part of the non-oxidative pentose phosphate pathway (PPP) and its deficiency results in many clinical symptoms including kidney and liver failure, which can lead to early child-mortality. Some of these patients suffer from recurrent infections, for example in the respiratory tract. Neutrophils are the most abundant white blood cells and essential for the innate immune defence against bacterial and fungal pathogens. The PPP generates reduced NADPH that is crucial for the generation of superoxide by the NADPH oxidase NOX2. In turn, NOX2 is essential for neutrophil extracellular trap (NET) formation. NETs occur through the neutrophil-specific cell death netosis and consist of chromatin decorated with granular proteins. Here I report that neutrophils of three TALDO patients did not make NETs. Deletion of transaldolase 1, and its partner enzyme transketolase, in the neutrophil-like PLB-985 cell line reduced ROS generation and cell death. This confirms that transaldolase 1 is required for NET formation. We present, to the best of our knowledge, the first genetic evidence that the non-oxidative PPP is required for ROS generation and NET formation. Furthermore, some of the metabolic requirements for NET formation were assessed. The obtained data indicate that the initial peak of the oxidative burst is irrelevant for NET formation but the ROS generation after 50 minutes on the contrary has crucial significance.

Tese de mestrado, Análises Clínicas, Universidade de Lisboa, Faculdade de Farmácia, 2013
The Pentose Phosphate Pathway (PPP) fulfils two unique functions: (i) the formation of ribose-5-phosphate (R5P) for the synthesis of nucleotides, RNA and DNA, supporting cell growth and proliferation; and (ii) the formation of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH). NADPH carries chemical energy in the form of reducing power, being essential to the cellular oxidative defense system and is also the universal reducing agent in many anabolic pathways, such as cholesterol and lipid synthesis and fatty acids chain elongation. The PPP comprises two separate branches: (i) an oxidative, non-reversible branch, and (ii) a non-oxidative, reversible branch. The oxidative branch primarily depends on glucose 6-phosphate dehydrogenase (G6PD), whereas transaldolase (TALDO) is the rate-limiting enzyme for the non-oxidative branch. Although the oxidative branch of the PPP is recognized as the source of NADPH and R5P, the overall contribution of the non-oxidative branch to metabolism and cell survival is poorly understood. Metabolism of glucose through the PPP influences the development of diverse pathologies. Deficiency of the PPP enzyme G6PD represents the most common genetic defect in humans and leads to NADPH depletion in red blood cells (RBC), predisposing to oxidative stress-induced hemolytic anemia. Recently, two new defects in the PPP were described: ribose-5-phosphate isomerase (RPI) deficiency and transaldolase (TALDO) deficiency. The first defect is associated with progressive leukoencephalopathy and mild peripheral neuropathy. The metabolic profile of the disease is associated with elevated levels of ribitol and arabitol in the brain, as showed by magnetic resonance spectroscopy (MRS), urine, plasma and cerebrospinal fluid (CSF). In contrast, TALDO deficiency is associated with liver symptoms, while other organs are affected to a variable degree. The metabolic phenotype of the disease is characterized by increased urinary concentrations of the polyols: erythritol, arabitol, ribitol, sedoheptitol, perseitol and the sugars: sedoheptulose and mannoheptulose, as well as sedoheptulose-7-phosphate. The profound influence of TALDO on NADPH levels, mitochondrial dysfunction and oxidative stress is ascribed to the blocked recycling of R5P to glucose 6-phosphate (G6P) and to the proposed linkage to aldose reductase (AR) that converts the accumulated C5-sugar phosphates to C5-polyols at the expense of NADPH. Mitochondrial dysfunction associated to TALDO deficiency leads to liver disease progressing from non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC). Therefore, TALDO deficiency can account for liver disease of variable severity. The diagnosis of the PPP deficiencies is possible by biochemical evaluation of polyols’ and sugars’ profiles in body fluids. Abnormal concentrations of these compounds occur in a number of pathological conditions such as diabetes mellitus, renal and liver disease and several inborn errors of carbohydrate metabolism, such as galactosemia. Therefore, it is necessary to take into account the variability of metabolic profiles and to understand their meaning. Along the thesis, it is our goal to revise the metabolic pathway of pentose phosphates and its implication in the associated disease states. Furthermore, the development and validation of the analytical methods (GC-FID and GC-MS) will be presented and discussed. We will also present our preliminary results upcoming from two separated studies, focused on the characterization of the PPP intermediate metabolites profile in (1) pre-selected cases with unexplained hepatosplenomegaly and/or liver dysfunction in a pediatric population; and (2) in adult patients with liver dysfunction (NAFLD or AFLD). We hope to find new biomarkers and metabolic signs which may open new perspectives of research for better understanding liver dysfunction. Interesting data is described, in the pediatric population, where a patient with an isolated sedoheptulokinase (SHPK) deficiency is reported. All the subsequent studies, some still in course, seem to confirm this new defect in the PPP. In the adult population our findings point to the putative importance of polyols as possible biomarkers of liver dysfunction progression.
A via das pentoses fosfato tem dois objectivos principais: (i) a formação de ribose-5-fosfato (R5P) para a síntese de nucleótidos, ARN e ADN, essenciais para o crescimento e proliferação celular; e (ii) a redução de nicotinamida adenina dinucleótido fosfato (NADPH). A NADPH transporta energia química sob a forma de poder redutor, sendo essencial para o sistema de defesa oxidativo das células e sendo ainda o agente redutor universal de inúmeras reacções anabólicas, tais como a síntese de colesterol e lípidos e o prolongamento das cadeias dos ácidos gordos. A via das pentoses fosfato é constituída por dois ramos: (i) um ramo oxidativo, não reversível, e (ii) um ramo não-oxidativo, completamente reversível. O ramo oxidativo é dependente da actividade da enzima glucose-6-fosfato desidrogenase (G6PD), enquanto a transaldolase (TALDO) é a enzima limitante do ramo não-oxidativo. Apesar do reconhecido papel do ramo oxidativo da via das pentoses fosfato como fonte de NADPH e R5P, a contribuição da via não-oxidativa no metabolismo e sobrevivência celular é pouco conhecida. O metabolismo da glucose na via das pentoses fosfato influencia o desenvolvimento de diversas patologias. A deficiência em G6PD é o defeito genético mais comum na população humana, levando a níveis reduzidos de NADPH nos eritrócitos e predispondo ao aparecimento de anemia hemolítica induzida por stress oxidativo. Recentemente, foram descritas duas novas deficiências enzimáticas na via das pentoses fosfato: a deficiência em ribose-5-fosfato isomerase (RPI) e a deficiência em transaldolase (TALDO). A primeira deficiência enzimática está associada a leucoencefalopatia progressiva e ligeira neuropatia periférica. O perfil metabólico da doença revela níveis elevados dos polióis: ribitol e arabitol no cérebro, tal como demonstrado por ressonância magnética, na urina, plasma e líquido cefalorraquidiano (LCR). Por outro lado, a deficiência em transaldolase está associada a alterações hepáticas, havendo um grau variado de comprometimento por parte de outros órgãos. O fenótipo metabólico desta deficiência está associado à excreção de níveis elevados dos polióis: eritritol, arabitol, ribitol, sedoheptitol, perseitol e dos açúcares: sedoheptulose e manoheptulose, bem como de sedoheptulose-7-fosfato. A forte influência da transaldolase nos níveis de NADPH em situações de disfunção mitocondrial e stress oxidativo é atribuída ao bloqueio da reciclagem da glucose-6-fosfato a partir da ribose-5-fosfato e à hipotética ligação à actividade de aldoses redutases (AR), responsáveis pela conversão dos açúcares-C5-fosfatados a C5- polióis, às custas de NADPH. A disfunção mitocondrial associada à deficiência em transaldolase leva a uma doença hepática progressiva que se inicia com o aparecimento do fígado gordo não alcoólico (NAFLD), passando a esteatohepatite não alcoólica (NASH), cirrose e carcinoma hepatocelular (CHC). Assim, a deficiência em transaldolase está associada a doença hepática de variado grau de severidade. O diagnóstico dos defeitos da via das pentoses fosfato é possível pela análise dos perfis bioquímico dos açúcares e polióis nos fluidos biológicos. Concentrações elevadas destes compostos aparecem em diversas situações patológicas, tais como a diabetes mellitus, a doença renal e hepática e vários erros hereditários do metabolismo dos hidratos de carbono, tais como a galactosemia. Assim, torna-se imperativo ter em conta a grande variabilidade de perfis metabólicos e o conhecimento dos seus significados. Ao longo desta tese, é nosso objectivo fazer uma revisão sistematizada da via das pentoses fosfato e a sua implicação em estados de doença. Será ainda apresentado e discutido o desenvolvimento e validação dos métodos cromatográficos (GC-FID e GC-MS) desenvolvidos. Finalmente, apresentaremos os resultados preliminares de dois estudos, iniciados com o objectivo de caracterizar os metabolitos intermediários da via das pentoses fosfato em (i) uma população pediátrica de casos pré-seleccionados com hepatoesplenomegália de causa desconhecida e/ou com disfunção hepática; e (ii) uma população de adultos com disfunção hepática (fígado gordo não alcoólico ou doença hepática devida ao abuso do consumo de álcool). Esperamos descobrir novos biomarcadores ou alterações metabólicas importantes, que possam abrir a porta a futuros estudos para melhor compreender a disfunção hepática. No estudo efectuado na população pediátrica são apresentados resultados interessantes de um doente em que se descreve pela primeira vez um défice isolado da enzima sedoheptulose cinase (SHPK). Todos os estudos subsequentes, alguns ainda em curso, apoiam a descoberta desta nova deficiência enzimática na via das pentoses fosfato. No estudo da população adulta com disfunção hepática, os nossos resultados apontam para uma possível importância do perfil dos polióis como biomarcadores da progressão da disfunção hepática. Palavras-chave: Via das pentoses fosfato; deficiência em ribose-5-fosfato isomerase; deficiência em transaldolase; fígado gordo não alcoólico; deficiência em sedoheptulose cinase.

Leung Ling Yan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (leaves 117-138).
Abstracts in English and Chinese.
Abstract (English) --- p.ii
Abstract (Chinese) --- p.iv
Acknowledgements --- p.vi
List of Abbreviation --- p.vii
List of Figures and Tables --- p.viii
Chapter Chapter 1 --- General Introduction --- p.1
Chapter Chapter 2 --- The effect of salinity on pentose phosphate pathway activity and red blood cells resistance to oxidative stress in silver seabream (Sparus sarba) --- p.4
Abstract --- p.7
Chapter 2.1 --- Literature review --- p.8
Chapter 2.2 --- Materials and Methods --- p.17
Chapter 2.3 --- Results --- p.32
Chapter 2.4 --- Discussion --- p.54
Chapter 2.5 --- Conclusion --- p.63
Chapter Chapter 3 --- The effect of dietary carbohydrate level on pentose phosphate pathway activity and red blood cells resistance to oxidative stress in of silver seabream (Sparus sarba) --- p.65
Abstract --- p.67
Chapter 3.1 --- Literature review --- p.68
Chapter 3.2 --- Materials and Methods --- p.77
Chapter 3.3 --- Results --- p.80
Chapter 3.4 --- Discussion --- p.101
Chapter 3.5 --- Conclusion --- p.112
Chapter Chapter 4 --- Summary --- p.113
References --- p.117

博士
高雄醫學大學
醫學研究所
98
Hepatocellular carcinoma ( HCC ) is the 5th most common cancer and is the third cause of cancer-related death in the world, which becomes a major global health problem. Now, the commonly used chemotherapies are accompanied by side effects and are easy to develop drug resistance. On the other hand, nutritional therapy has become an alternative choice for the treatment of cancer and shown a good effect. Arginine and vitamin C are essential nutrients for human and they function as a vasodilator and antioxidant, at the physiological concentration ranges. However, it is still not known whether they are suitable to be components of the formular of nutritional therapy or not. This study was aimed to test the possibility of arginine and/or vitamin C treatment on the malignant hepatoma cell line ( HA22T/VGH ). The molecular mechanism would be further explored. Cell viability assay by trypan blue exclusion method showed that arginine at the concentration of 1,000 μg/ml or vitamin C 100 μg/ml was not toxic to HA22T/VGH cells. However combination of both reagents were toxic and be the IC50 concentration. Furthermore, we found this combination induced apoptosis by activation of the mitochondrial apoptosis-related proteins such as Bax, cytochrome c, and caspase-9, and by inhibition of anti-apoptotic proteins of Bcl-2 and Bcl-xL. In addition, the death receptor of FasL, Fas, FADD, and caspasse-8 were also activated. Importantly, activation of the critical caspase-3 caused DNA fragmentation. Interesting, we found this combination-induced apoptosis would be reversed by the caspase-8, -9, -3 and caspase-family inhibitor. The flow cytometry analysis showed arginine or vitamin C did not affect the levels of reactive oxygen species ( ROS ) and reactive nitrogen species ( RNS ), while combined treatment could significantly increased ROS and RNS and change mitochondrial membrane potential. Furthermore, the intracellular ATP and glutathione levels are also decreased. The key enzymes of the pentose phosphate pathway ( PPP ) including glucose 6-phosphate dehydrogenase ( G6PD ), 6-phospho-gluconate dehydrogenase ( 6PGD ), and transketolase ( TAL ) activity were also decreased by this combination treatment. Due to the decrease in NADPH, the GSH homeostasis could not be maintained. Moreover, addition of the antioxidants of glutathione, catalase, or N-acetyl-L-cysteine ( NAC ), or glucose-6-phosphate, pyruvate or nitric oxide synthase inhibitor ( L-NAME ) could significantly inhibit this combination-induced apoptosis. Noticeably, addition of glutathione synthesis inhibitor ( L-buthionine-[S,R]-sulfoximine ; BSO ) which markedly increased the levels of ROS and RNS, elevated about 25 % apoptotic cell death. These finding demostrated that combination treatment of arginine and vitamin C could induce apoptosis of HA22T/VGH through the pentose phosphate pathway and reduce NADPH synthesis resulting in interference in the cellular redox state. The study provides the potential nutrient therapy of combined arginine and vitamin C treatment in hepatoma and the molecular mechanisms were also revealed.

Apoptosis is a form of programmed cellular "suicide" which is activated in response to a variety of pro-death stimuli. Apoptotic cell death is orderly and energy-dependent, and cellular constituents are packaged into membrane-bound vesicles for consumption by phagocytes. Toxic intracellular signals are never exposed to neighboring cells or to the extracellular environment, and a host inflammatory response does not occur. Apoptosis is executed by the coordinated activation of caspase family proteins. Caspase-2 is an apical protease in this family, and promotes cell death after receipt of cues from intracellular stressor signals. Caspase-2 helps to initiate apoptosis by responding to cellular death stimuli and signaling for downstream cytochrome c release and executioner caspase activation.

Several years ago our lab determined that Xenopus laevis oocyte death is partly controlled by the activation of caspase-2. In the setting of oocyte or egg extract nutrient depletion, caspase-2 was observed to be activated upstream of mitochondrial cytochrome c. In fact, caspase-2 is suppressed in response to the nutrient status of the oocyte: nutrient-replete oocytes with healthy pentose phosphate pathway flux and abundant NADPH production are able to inhibit caspase-2 via S135 phosphorylation catalyzed by calcium/calmodulin-dependent protein kinase II. Phosphorylation of caspase-2 at S135 is critical in preventing oocyte cell death, and a caspase-2 mutant unable to be phosphorylated loses its ability to respond to suppressive NADPH signals.

In this dissertation we examine the converse mechanism of metabolically-regulated caspase-2 activation in the Xenopus egg extract. We now show that caspase-2 phosphorylated at S135 binds the interactor 14-3-3 zeta, thus preventing caspase-2 dephosphorylation. Moreover, we determined that S135 dephosphorylation is catalyzed by protein phosphatase-1, which directly binds caspase-2. Although caspase-2 dephosphorylation is responsive to metabolism, neither PP1 activity nor binding is metabolically regulated. Rather, release of 14-3-3 zeta from caspase-2 is the point of metabolic control and allows for caspase-2 dephosphorylation. Accordingly, a caspase-2 mutant unable to bind 14-3-3 zeta is highly susceptible to activation. Although this mechanism was initially established in Xenopus, we now demonstrate similar control of murine caspase-2 by phosphorylation and 14-3-3 binding in mouse eggs.

In the second part of this dissertation we examine the paradigm of caspase-2 metabolic regulation in a mammalian somatic cell context. We observed that mammalian caspase-2 is a metabolically-regulated phosphoprotein in somatic cells, and that the site of regulation is caspase-2 S164. Phosphorylation at S164 appears to inhibit mammalian caspase-2 by preventing its induced proximity oligomerization, thus also preventing procaspase-2 autocatalytic processing. We further identify some of the molecular machinery involved in S164 phosphorylation and demonstrate conservation with the validated Xenopus regulators. Interestingly, we extend the findings of caspase-2 phosphorylation to a study of ovarian cancer, and show that caspase-2 S164 phosphorylation might be involved in determining cancer cell chemosensitivity. We further provide evidence that chemosensitivity can be modulated by the cellular metabolic status in a caspase-2-dependent manner. Thus, we have identified a novel phosphorylation site on mammalian caspase-2 in somatic cells, and are working further to understand the implications of caspase-2 signaling in the context of cancer cell responsiveness to chemotherapeutic treatments.

Dissertation

Soybean (Glycine max (L.) Merr) is the largest oil and protein crop in the world and it is grown for both oil and protein. To address the needs of both the edible oil market and industrial applications of soybean oil, fatty acid modification has been a focus of soybean breeding programs. Natural variation, mutagenesis and genetic engineering have been used to alter the fatty acid profile. Several genes, mostly desaturases, have been associated with altered fatty acid profiles but enzymes in the Kennedy Pathway have yet to be studied as another source of genetic variation for altering the fatty acid profiles. The Kennedy Pathway is also known as the oil producing pathway and consists of four enzymes: glycerol-3-phosphate acyltransferase (G3PAT); lysophosphatidic acid acyltransferase (LPAAT); phosphatidic acid phosphatase (PAP); and diacylglycerol acyltransferase 1 (DGAT1). The starting material for this pathway is glycerol-3-phosphate, which is produced from glycerol by glycerol kinase (GK), and the product of this pathway is triacylglycerol (TAG). The overall objective of this study was to elucidate the role that the Kennedy Pathway plays in determining the fatty acid profile in two ways: (1) sequencing the transcribed region of the genomic genes encoding the enzymes of GK, G3PAT, LPAAT, and DGAT1 in soybean genotypes with altered fatty acid profiles; and (2) studying their expression over seed development, across three growing temperatures. The genetic material for the study consisted of four soybean genotypes with altered fatty acid profile: RG2, RG7, RG10, and SV64-53. Results from sequencing showed that the mutations identified in G3PAT, LPAAT, and DGAT1 in the four soybean genotypes did not explain the differences in the fatty acid profiles. The expression of G3PAT, LPAAT, and DGAT1 over seed development showed that G3PAT had the lowest levels, followed by LPAAT, then DGAT1, across the growing temperatures. The differences in expression among genotypes corresponded to differences in fatty acid accumulation, suggesting that expression rather than genetic mutations in the transcribed region of the genes influenced the fatty acid profile of the genotypes in this study. In conclusion, the enzymes of the Kennedy Pathway appear to contribute to the altered fatty acid profiles observed in the soybean mutant genotypes.
Ontario Ministry of Economic Development and Innovation (formerly Ontario Ministry of Research and Innovation), BioCar Initiative, Grain Farmers of Ontario, SeCan

Liquid chromatography tandem mass spectrometry has become a powerful tool for investigating biological systems. Herein we describe the development of both isotope dilution mass spectrometry methods and targeted metabolomics methods for the study of metabolic and cell-cell signaling applications. A putative yeast enzyme was characterized by discovery metabolite profiling, kinetic flux profiling, transcriptomics and structural biology. These experiments demonstrated that the enzyme shb17 was a sedoheptulose bisphosphatase that provides a thermodynamically dedicated step towards riboneogenesis, leading to the redefinition of the canonical pentose phosphate pathway. An extension of metabolic profiling and kinetic flux profiling methods was developed for a set of symbiotic marine microorganisms. Carbon flux from the most abundant photosynthetic organism, Prochlorococcus, to a symbiotic Alteromonas was observed in liquid coculture. These methods enable a more biologically relevant assay for marine species and will lead to a better understanding of carbon flux in the oceans. Energy taxis refers to the active migration of bacteria in response to electron transport system related signals. The second messenger cyclic-di-GMP provides a link between the metabolic signals and motility. Quantitation of c-di-GMP helped characterize the nature of this regulation. Autoinducer-2 is a small sugar produced by a large variety of bacteria that is proposed to be a universal quorum sensing signal. The quorum sensing function of autoinducer-2 is disputed because it is produced by an enzyme of the activated methyl cycle, leading to an alternate hypothesis that it is simply a metabolic byproduct. Herein a method for the detection of autoinducer-2 is developed to enable studies of its signaling role and biosynthetic regulation. These studies demonstrated that autoinducer-2 does not function as a signal in all species. Further, metabolic experiments indicated that the metabolic impact of LuxS dysfunction was small and could be mitigated by recycling oxidized glutathione. Together, these data indicate that neither hypothesis is adequate. Evidence is provided that autoinducer-2 suppresses the immune system, by the interruption of cytokine signaling, implying that autoinducer play a protective role during host colonization.

Content removed due to copyright restrictions: Webby, C.J., Patchett, M.L. & Parker, E.J. (2005) Characterization of a recombinant type II 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Helicobacter pylori. Biochemical Journal 390, 223-230 Webby C.J., Lott J.S., Baker H.M., Baker E.N., & Parker E.J. (2005) Crystallization and preliminary X-ray crystallographic analysis of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Mycobacterium tuberculosis. Acta Crystallographica Section F - Sturctural Biology and Crystallization Communications 61(4) 403-406. Webby C.J., Baker H.M., Lott J.S., Baker E.N. & Parker E.J. (2005) The structure of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Mycobacterium tuberculosis reveals a common catalytic scaffold and ancestry for type I and type II enzymes. Journal of Molecular Biology 354(4), 927-939
The shikimate pathway, responsible for the biosynthesis of aromatic compounds, is found in microorganisms and plants but absent in higher organisms. This makes the enzymes of this pathway attractive as targets for the development of antibiotics and herbicides. Recent gene disruption studies have shown that the operation of the shikimate pathway is essential for the viability of M. tuberculosis, validating the choice of enzymes from this pathway as targets for the development of novel anti-TB drugs. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first committed step of the shikimate pathway. Two distinct classes of DAH7PS have been defined based on sequence similarity. The type I DAH7PSs are well characterized, however prior to this project there was limited mechanistic and no structural information about type II enzymes. Sequence identity between type I and type II enzymes is less than 10% raising the possibility that they represent distinct protein families, unrelated by evolution. We have functionally characterized the type II enzyme from Helicobacter pylori, and have shown that type I and type II enzymes catalyze a metal-dependent ordered sequential reaction following the same stereochemical course. We have solved the structure of the type II DAH7PS from M. tuberculosis using single-wavelength anomalous diffraction (SAD) methods and the structure reveals a tightly associated dimer of (β/α)8 TIM barrels. The monomer fold, the arrangement of key residues in the active site, and the binding modes of PEP and Mn2+, all match those of the type I enzymes. This similarity of protein fold and catalytic architecture makes it unequivocal that type I and type II enzymes are related by divergent evolution from a common ancestor. Interestingly, there are significant differences in the additional structural elements that extend from the core (β/α)8 barrel and in the quaternary structure. Further structural and functional analysis of M. tuberculosis DAH7PS revealed that the two major additions decorating the barrel are involved in the binding of the aromatic amino acids. Two distinct inhibitory binding sites for Trp and Phe have been identified providing an explanation for the synergistic inhibition displayed with Trp and Phe. The role of several active site residues of Mt-DAH7PS in enzyme catalysis has also been investigated.

Ce projet a pour but d’évaluer la capacité de la voie des pentoses phosphates (VPP) dans les racines transgéniques de pomme de terre (Solanum tuberosum) modifiées pour exprimer différents niveaux de l'hexokinase (HK) et de la triosephosphate isomérase cytosolique (cTPI). Dans les racines, la VPP alimente la voie de l’assimilation de l’azote en equivalents réducteurs et permet donc la biosynthèse des acides aminés. Le glucose-6-phosphate produit par l’HK est consommé par la partie oxydative de la VPP catalysée par la glucose-6-phosphate déshydrogénase (G6PDH) et la 6-phosphogluconate déshydrogénase (6PGDH). Les changements dans l'expression de HK et cTPI peuvent affecter le fonctionnement de la VPP et les mécanismes qui sont liés à l’utilisation des équivalents réducteurs produits par la VPP, comme l'assimilation de l’azote et la synthèse des acides aminés. Afin d’évaluer l’effet des manipulations génétiques de l’HK et de la cTPI sur l’assimilation de l’azote, nous avons cultivé les racines transgéniques sur des milieux contenant des concentrations élevées (7 mM) ou basses (0,7 mM) de nitrate d’ammonium comme source d’azote. Les résultats montrent que la culture sur un milieu riche en azote induit les activités G6PDH et 6PGDH. Les données montrent que la capacité de la VPP est plus grande avec des niveaux élevés en HK ou en cTPI. Nous avons aussi pu démontrer une plus grande activité spécifique de l’HK dans les conditions pauvres en azote. Ces données ont été complémentées par des mesures des pools d’acides aminés dans les racines transgéniques cultivées sur différents niveaux d’azote. Aucune tendance notable des pools d’acides aminés n’a été remarquée dans les racines modifiées pour leur contenu en HK suggèrant que la manipulation de HK n’affecte pas l'assimilation de l’azote. Dans les racines transgéniques modifiées pour la cTPI, les ratios Gln/Glu et Asn/Asp sont plus élevés chez les clones antisens, indiquant une assimilation de l’azote plus élevée. Ces résultats ont démontré l'activation de l'assimilation de l’azote chez les clones antisens cTPI dans les conditions élevées et basses d’azote alors que la manipulation de l’HK n’affecte pas l’assimilation de l’azote.
This study investigates the capacity of the oxidative pentose phosphate pathway (oxPPP) and nitrogen metabolism in transgenic potato (Solanum tuberosum) roots modified to express different levels of hexokinase (HK) or cytosolic triosephosphate isomerase (cTPI) growing under different nitrogen regimes. The flux of carbon through the oxPPP in cTPI antisense roots is higher than control roots growing under high supply of N. On the other hand, the conversion of Glucose (Glc) to Glucose-6-phosphate (G6P) is higher in roots overexpressing HK than in antisense HK roots growing at a high level of N. Therefore, overexpression of HK or down regulation of cTPI activities in transgenic roots might be compensated by increased C catabolism through the oxPPP. In order to see the affect of HK and cTPI manipulation on N assimilation, the transgenic roots were grown on media with low or high concentration of ammonium nitrate as the N source. The specific activity of the oxPPP enzymes glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) were both increased by an increased N supply in HK and cTPI transgenic roots. This is consistent with the provision of reducing equivalents for N assimilation. The data also show that the capacity of the oxPPP is higher in roots with high HK or cTPI activity. We were able to detect higher HK specific activity in N deficient conditions. These data were complemented with measurements of amino acid pools in transgenic roots. No trend in amino acid pools was found in roots modified for HK activity. However, down regulation of cTPI led to higher Gln, Gln/Glu and Asn/Asp ratios, indicating higher assimilation of N. These results demonstrated the activation of N assimilation in cTPI antisense clones while the manipulation of HK is unlikely to affect the N assimilation.