Dissertationen zum Thema „Hypoxic translation“

Identifying exploitable differences between cancer cells and normal cells has been ongoing since the dawn of cancer therapeutics. This task has proven difficult due to the complex genetic makeup of cancers. Tumours, however, share a low oxygen (hypoxic) microenvironment that selects for malignant cancer cells. It has recently been shown that cells switch from eIF4E to eIF4E2-mediated protein synthesis during periods of hypoxia, similar to those found in tumour cores. We hypothesize that this hypoxic translation complex is required for cell survival in hypoxia and can be targeted by inhibiting the eIF4E2 cap-binding protein. Here, we show that genetically diverse cancer cells require the cap-binding protein eIF4E2 for their growth, proliferation, and resistance to apoptosis in hypoxia, but not in normoxia. Furthermore, in vitro and in vivo eIF4E2-depleted tumour models cannot grow or sustain hypoxic regions without the reintroduction of exogenous eIF4E2. Thus, tumour cells could be targeted over somatic cells by selectively inhibiting their protein synthesis machinery, much like the function of antibiotics that revolutionized medicine.

Multiple sclerosis is a chronic inflammatory disease of the CNS associated with widespread primary demyelination and axonal degeneration. The mechanisms underlying the expression of neurological deficits are incompletely understood. Recent histological findings are consistent with a view that active MS lesions may be hypoxic (i.e. suffer a low oxygen concentration). For example, inflammatory ‘Pattern III’ MS lesions express hypoxia inducible factor-1a (HIF-1a), a master regulator of genes whose function is to bias a cell for survival under hypoxic conditions. Hypoxia also results in a number of other consequences designed to limit energy expenditure, including the inhibition of protein translation by the phosphorylation of the eukaryotic initiation factor (eIF-2a), and the formation of stress granules (SGs). Both hypoxia and the inhibition of protein synthesis could cause neurological deficits and thus contribute to the neurological deficits of MS. The aim of the present study is to explore experimental models of MS for evidence of a) hypoxia and b) inhibition of protein translation. The experimental models of inflammatory demyelinating lesions were induced either by the intraspinal injection of lipopolysaccharide (LPS) into the rat dorsal column (LPS-DC, focal lesion), or by immunization of rats or mice to induce experimental autoimmune encephalomyelitis (EAE, disseminated lesions). Animals were examined daily for the expression of any neurological deficit, and tissue hypoxia was detected during life by the systemic administration of pimonidazole, a marker for hypoxia, several hours before termination. Tissue was taken at different stages of lesion development (1-28 days post injection) and examined immunohistochemically for the presence of hypoxia, determined by the expression of binding for pimonidazole, and for the inhibition of protein translation by examining the expression of eIF-2a and SGs. Other markers of disease activity were also examined, including a marker of microglial/macrophage activation (ED-1), HIF-1a, and glucose transporter-1 (GLUT-1). In LPS lesions, labelling for pimonidazole was most intense at the site of injection, 24 hours later. In EAE, labelling for pimonidazole was present as early as 2 days post immunization, but it was expressed more strongly when animals were exhibiting a neurological deficit, subsiding thereafter. In animals injected with LPS, eIF-2a and SGs were expressed most intensely 24 hours post LPS injection, localised to the spinal motor neurons. In EAE, eIF-2a and SGs were expressed in spinal motor neurons, and in cerebellar neurons, at the onset of neurological deficits. These findings reveal for the first time that inflammatory demyelinating lesions are associated with the presence of tissue hypoxia and markers of the inhibition of protein synthesis. It appears that these phenomena may contribute to the expression of neurological deficits, opening new opportunities for therapy.

Adaptation to variations in oxygen concentration is a conserved mechanism in all metazoans as this process is central for the maintenance of cell and tissue homeostasis. Two major and highly conserved processes contribute to hypoxia-induced gene reprogramming. The first one relies on the transcriptional activation of gene expression by the Hypoxia Inducible Factors (HIFs) leading to the upregulation of a large panel of genes. The second one corresponds to a strong modification of the translation program. While the mechanisms underlying HIF-dependent transcriptional activation have been well characterized, the ones governing translation reprogramming are only partially understood. 
To uncover how mRNA translation takes place at low oxygen tension, we used Drosophila as our research model since both Drosophila flies and S2 cells are highly resistant to low O2. We first demonstrate that several genes are efficiently translated under hypoxia in Drosophila S2 cells. By a gene reporter-based approach, we demonstrate that Ldh mRNA 3’UTR is sufficient to promote reporter mRNA association to polysomes in hypoxia. A deletion analysis of Ldh 3’UTR leads to the identification of a ACAAA-rich sequence important for polysomal association and translation in hypoxia.Cap-binding factors play a key role in controlling translational initiation. We have shown that the cap-binding translation initiation factor eIF4EHP (4EHP) plays a dual role on translation under hypoxic conditions. Despite having a general repressive function on global translation under normoxic and hypoxic conditions, we demonstrated that 4EHP also positively controls the translation of specific mRNAs under hypoxia. Inactivation of 4ehp reduces LDH protein synthesis and impairs reporter mRNA translation in hypoxia. Deletion of 4ehp inhibites the translation of several candidate genes harboring a ACAAA motif in 3’UTR under hypoxia, suggesting that 4EHP is required for hypoxic translation of mRNAs carrying ACAAA-rich motifs. Most interestingly, we observed that 4EHP is strongly enriched in polysomal fractions in hypoxia, further supporting a role of this initiation factor in hypoxic translation. The reduction of 4ehp expression also impairs Drosophila development under hypoxic conditions. Taken together, our results indicate that specific mRNAs can bypass the translational blockade imposed by hypoxic conditions. This process is controlled by mRNA 3’UTR “CA” rich element and is positively regulated by the translation initiation factor 4EHP.
L'adaptation aux variations de la concentration en oxygène est un mécanisme conservé chez tous les métazoaires car ce processus est central pour le maintien de l'homéostasie cellulaire et tissulaire. Deux processus hautement conservés contribuent à la reprogrammation génétique induite par l'hypoxie. Le premier repose sur l'activation transcriptionnelle de l'expression génique par les facteurs inductibles de l'hypoxie (HIF) conduisant à l’induction d'un large panel de gènes. Le second correspond à une forte modification du programme traductionnel. Alors que les mécanismes sous-jacents à l'activation transcriptionnelle dépendante de HIF ont été bien caractérisés, ceux qui régissent la reprogrammation de la traduction ne sont que partiellement compris.Pour découvrir comment la traduction de l'ARNm se déroule à faible tension d'oxygène, nous avons utilisé la drosophile comme modèle d’étude, car les mouches Drosophila melanogaster et les cellules S2 issue de cet organisme sont très résistantes à de faibles teneurs en O2. Nous avons tout d’abord démontré que plusieurs gènes sont efficacement traduits en hypoxie dans les cellules S2 de drosophile. Par une approche basée sur l’utilisation de gènes rapporteurs, nous avons démontré que la région 3’ Non traduite (3’UTR) de l’ARNm Ldh est suffisante pour promouvoir l’association de l’ARNm du rapporteur aux polysomes en conditions hypoxiques. Une analyse par délétion de la région 3’UTR de l’ARNm Ldh a conduit à l’identification d’une séquence riche en ACAAA importante pour l’association polysomale et la traduction en hypoxie. La reconnaissance de la coiffe joue un rôle clé dans le contrôle de l'initiation de la traduction Nous avons montré que le facteur d'initiation de la traduction eIF4EHP (4EHP), qui se lie à la coiffe, joue un double rôle sur la traduction dans des conditions hypoxiques. Bien qu'il ait une fonction répressive sur la traduction générale dans des conditions normoxiques et hypoxiques, nous avons démontré que 4EHP contrôle aussi positivement la traduction d'ARNm spécifiques dans des conditions hypoxiques. L'inactivation de 4ehp réduit la synthèse de la protéine LDH et altère la traduction de l'ARNm rapporteur contenant la partir 3’UTR du messager Ldh en hypoxie. La délétion de 4ehp peut atténuer la traduction de plusieurs gènes candidats contenant un motif ACAAA dans leur région 3’UTR 3' en hypoxie, ce qui suggère que 4EHP est nécessaire pour la traduction hypoxique des ARNm portant des motifs riches en ACAAA. De façon intéressante, nous avons observé que 4EHP est fortement enrichi dans les fractions polysomales en hypoxie, ce qui confirme le rôle de ce facteur d'initiation dans la traduction en hypoxie. La réduction de l'expression de 4ehp altère également le développement de la Drosophile dans des conditions hypoxiques. Ensemble, nos résultats indiquent que des ARNm spécifiques peuvent contourner le blocage traductionnel imposé par les conditions hypoxiques. Ce processus est contrôlé par l'élément riche en "CA" situé dans la partie 3'UTR de l’ARNm et est régulé positivement par le facteur d'initiation de la traduction 4EHP.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Introduction: Persistent pulmonary hypertension of the newborn (PPHN) is associated with an elevated thromboxane to prostacyclin ratio, pulmonary artery (PA) hyperreactivity and hypersensitivity. Thromboxane receptor (TP), coupling with G-protein Gαq causes pulmonary vasoconstriction; whereas prostacyclin receptor (IP), coupling with Gαs, causes vasodilation and TP phosphorylation via adenylyl cyclase (AC)-cAMP-protein kinase A (PKA), desensitizes TP. Both TP phosphorylation and Gαq palmitoylation play major roles in regulation of signaling through the TP-Gαq complex. We hypothesized that increased Gαq palmitoylation and decreased AC activity could cause hypoxic TP hyperresponsiveness. We studied the impact of hypoxia on selected post-translational modifications of the receptor-G-protein complex, determining TP vasoconstriction: Gαq palmitoylation, TP phosphorylation and upstream AC activity. Methods: Force responses to thromboxane mimetic U46619, palmitoylation inhibition by 2-bromopalmitate (2-BP) and AC activation (forskolin) were studied by myography in hypoxic PPHN and control newborn swine pulmonary artery. Ca2+ mobilization was studied by fluorescent calcium indicators fura-2AM in pulmonary myocytes (PASMC), and fluo-4NW in HEK293 cells. Effects of hypoxia on Gαq palmitoylation were studied by metabolic labeling. Gαq cysteines and TP serines were mutated to determine sites of post-translational modifications. Protein expression and receptor-G-protein coupling were studied by Western blot and co-immunoprecipitation. PKA activity was assayed; and AC activity quantified. Results: Hypoxia increases Gαq palmitoylation, without increasing total palmitate uptake. Palmitoylation inhibition decreases U46619-stimulated force generation as well as Ca2+ mobilization in PPHN PA rings and hypoxic PASMC. Mutation of palmitoylable cysteine and palmitoylation inhibition proportionately decrease U46619-mediated Ca2+ mobilization in HEK293 cells. TP serine phosphorylation is decreased by hypoxia due to decreased PKA activity; this causes TP hypersensitivity and hyper-reactivity. Serine 324 of TPα is the target of PKA-mediated desensitization. AC activator-induced relaxation is reduced in PPHN PA. Basal and receptor-stimulated AC activity are decreased in hypoxic PASMC. Decreased AC activity is not due to decreased AC expression, ATP availability nor increased Gαi activation. Conclusion: Increased Gαq palmitoylation plays a role in TPα hyper-responsiveness in hypoxic PPHN. Hypoxia also reduces responses to agents acting through AC, unleashing TP-mediated vasoconstriction. Reactivation of pulmonary AC might be useful therapeutically to promote vasodilation and TP desensitization.
October 2016

Au cours de mon travail de thèse j’ai étudié la relation entre les facteurs participant à la terminaison de la traduction et ceux participant à la dégradation des ARNm chez S. cerevisiae.D’une part, je me suis intéressée au facteur Tpa1, caractérisé pour son rôle dans la terminaison de la traduction et la stabilité des ARNm chez S. cerevisiae et dont l’homologue chez S. pombe, Ofd1, participe au contrôle de la réponse hypoxique. Je me suis basée sur la structure de ce facteur, établie par nos collaborateurs pour comprendre plus précisément la fonction moléculaire de Tpa1 et rechercher des similitudes avec sa fonction chez S. pombe.Tpa1 est composée de deux domaines de type DSBH dont le premier, contenant le site catalytique, présente des homologies structurales avec la famille des prolyl-hydroxylases.Nous avons reproduit l’effet de la protéine Tpa1 sur la translecture in vivo et montré que son site catalytique prédit, ainsi que la présence des deux domaines étaient nécessaires pour cette activité. Nous avons aussi observé que Tpa1 inhibait par un mécanisme inconnu le facteur de transcription Hap1, qui régule des gènes en fonction de la quantité d’oxygène. Basé sur l’existence d’un inhibiteur d’Ofd1 chez S. pombe, nous avons ensuite montré qu’Ett1 (l’homologue de cet inhibiteur chez S. cerevisiae) avait un rôle similaire à Tpa1 dans la translecture. Une étude structurale collaborative d’Ett1 a mis en évidence une région conservée, se liant à une molécule de sulfate et à un ligand inconnu. Cette région est importante pour la translecture. Cependant, le substrat de Tpa1 reste pour l’instant inconnu comme les rôles précis de Tpa1 et Ett1 dans la terminaison de la traduction et dans la réponse à l’hypoxie.D’autre part, j’ai étudié le processus de NMD, particulièrement en me focalisant sur le mécanisme de discrimination entre un codon stop précoce (PTC) et un codon stop normal, et en analysant également la modification post-traductionnelle d’un facteur central du NMD, Upf1. Nous avons mis en évidence, qu’en plus de la région aval, la région en amont du PTCparticipait à sa reconnaissance. Nous avons testé plusieurs hypothèses sur le rôle de cette région, qui ont confirmé son rôle sans permettre de démontrer un mécanisme définitif. En parallèle, l’étude de la protéine Upf1 s’est concentrée sur ses modifications posttraductionnelles, particulièrement par phosphorylation. En effet, une telle modification est importante chez son homologue humain. Nous avons pu confirmer l’existence d’une forme modifiée et démontrer que celle-ci était localisée entre les acides aminés 153 et 971. Cette modification s’est avérée être très labile ce qui n’a pas permis de confirmer qu’il s’agissait d’une phosphorylation, ni de la cartographier plus précisément
During my PhD thesis, I analyzed the relation between factors that participate intranslation termination and those participating in mRNA decay in yeast S. cerevisiae.First, I focused on Tpa1, that had been proposed to participate in translationtermination and mRNA decay in S. cerevisiae, and whose homologue in S. pombe, Ofd1,participates to the control of hypoxic response. Based on the structure of Tpa1, established byour collaborators, I performed functional analysis to understand more precisely the molecularfunction of Tpa1 and similarities with its role in S. pombe. Tpa1 is composed of two DSBHdomains; the first, which contains the catalytic site, has structural homologies with the familyof prolyl-hydroxylase. We could reproduce the effect of Tpa1 on stop codon readthrough invivo and we showed that the predicted catalytic site and the presence of the two domains ofTpa1 were necessary for its activity. We also showed that Tpa1 inhibited one factor, Hap1,implicated in regulation of gene expression by oxygen. The existence of an inhibitor of Ofd1in S. pombe, allowed the identification of Ett1 (its homologue in S. cerevisiae). We showedthat Ett1 has a role similar to the one of Tpa1 in translational readthrough. A collaborativestructural and functional study of Ett1 revealed a conserved region, which binds a sulfate ion,and an unknown ligand. This region is important for the readthrough. However, thesubstrate(s) of Tpa1 remain(s) for the moment unknown, and the precise roles of Tpa1 andEtt1 in translation termination and in response to hypoxia remain to be deciphered.I also analyzed the NMD process by focusing more particularly on the mechanism thatallows the discrimination between a normal stop and a PTC (premature termination codon)and on the analysis of the post-translational modification of an important factor for the NMD,Upf1. This study revealed that, not only the region downstream of the PTC but also theupstream region participates to its recognition. We have tested several hypotheses on the roleof this upstream region, which confirmed its implication but did not reveal a definitivemechanism. In parallel, we started the study of the post-translational modifications of Upf1,and more particularly by phosphorylation. Indeed, the phosphorylation of Upf1 in human isvery important for the NMD process. We could confirm the presence of a modified form ofyeast Upf1 and we have demonstrated that it was localized between amino acids 153 and 971.This modification appeared to be highly labile. This prevented us to confirm definitively thatit was really a phosphorylation and to cartography precisely its location

Vascular disease is the leading cause of mortality and morbidity in the western world, and account for the 1 of every 3 death?s in the US, but a cure for vascular disease is yet to be realized. Hematopoietic stem progenitor cells (HSPCs) are mobilized from bone marrow and have the innate propensity to accelerate vascular repair by reendothelialization and revascularization of ischemic areas. The vasoreparative ability of HSPCs is largely due to their capacity to home to the areas of hypoxia and their sensitivity to hypoxia plays a critical role in the vasoreparative functions of these cells. The discovery of vasoreparative potential of HSPCs resulted in a breakthrough approach of cell-based therapies for the treatment of ischemic vascular diseases. However, success of this approach is essentially dependent on the number of cells that could be collected from an individual. Therefore, novel mechanism-based strategies are needed to enhance the outcomes of autologous cell-based therapies in poor mobilizers and older adults. Recent evidence of a potential role of the vasoprotective axis of the renin angiotensin system (RAS) in HSPCs functions offers a breakthrough. Angiotensin-(1-7), the primary mediator of the protective functions which acts on Mas receptor (MasR), is generated by angiotensin converting enzyme-2 (ACE2). In this study, we tested the effects of hypoxia on stimulation of vasoreparative potential of HSPCs and in upregulation of ACE2 and MasR. Importantly, we delineated the molecular mechanism of hypoxic exposure in regulation of ACE2 and MasR in a HIF1?- dependent manner and hypoxic exposure induced shedding of the membrane bound ACE2 in HSPCs. We used luciferase, a reporter assay, cell-based assays, gene/protein expression studies and pharmacological strategies in human and mouse HSPCs to test our hypotheses. To verify the biological significance of hypoxia, we performed in vivo studies in mice and humans, which recapitulated the in vitro observations on vascular protective axis of RAS in HSPCs. Collectively, these studies provided mechanistic insights into hypoxic regulation of vascular protective axis of RAS in HSPCs and also provided compelling evidence for the clinical use of hypoxia as a promising approach for enhancing the vasoreparative outcomes of cell-based therapies.
American Heart Association grant, 13SDG16960025
National Institutes of Health, National institute of Aging (NIA), 1R01AG056881

La traduction est une étape de l'expression des gènes fortement régulée. Lorsque la cellule est stressée, cela bloque la synthèse protéique globale tout en activant la traduction de certains ARNm par des mécanismes alternatifs. L'un de ces mécanismes implique des structures de l'ARNm, les IRES (Internal Ribosome Entry Sites), qui permettent un recrutement de la machinerie de traduction indépendamment de l'extrémité 5' coiffée de l'ARNm. L'activité des IRES est régulée par des facteurs appelés ITAF (IRES trans-acting factor). Le stress hypoxique survient dans différentes pathologies comme l'ischémie cardiaque et le cancer. Pour répondre rapidement à ce stress, les cellules produisent des facteurs de croissance (lymph)angiogéniques qui stimulent la formation de vaisseaux sanguins et lymphatiques. Cela permet de reperfuser la zone lésée dans le cœur ischémique, ou de stimuler la croissance tumorale et la dissémination métastatique dans le cancer. Mon projet de thèse porte sur l'identification d'ITAF contrôlant la traduction des ARNm de ces facteurs de croissance lors de l'hypoxie. La première partie de ma thèse s'est focalisée sur la régulation de la traduction dans les cardiomyocytes hypoxiques. Une étude semi-globale a montré que les gènes de la (lymph)angiogenèse sont régulés majoritairement au niveau traductionnel, alors que tous les ARNm connus pour posséder des IRES sont recrutés plus activement dans les polysomes lors du stress. Nous avons montré que les IRES d'ARNm des familles FGF (fibroblast growth factor) et VEGF (vascular endothelial growth factor) sont tous activés lors de l'hypoxie précoce alors que les IRES d'ARNm non liés à la (lymph)angiogenèse sont activés plus tardivement. Enfin, nous avons identifié un nouvel ITAF, la vasohibine (VASH1), qui active spécifiquement l'IRES du FGF1, mais pas les autres IRES testés. Une analyse par PCR array indique cependant que VASH1 est capable d'inhiber ou de stimuler la traduction de nombreux autres ARNm. La recherche d'un mécanisme global d'activation des IRES des FGF et VEGF fait l'objet du deuxième chapitre de ma thèse. VASH1 n'étant pas un ITAF commun, j'ai recherché d'autres candidats. Je suis partie de l'observation qu'un autre ITAF identifié précédemment au laboratoire, p54nrb, est un composant essentiel du paraspeckle, un corps nucléaire formé en cas de stress. Nous avons émis l'hypothèse que d'autres composants du paraspeckle pourraient être des ITAFs, en particulier le long ARN non codant NEAT1 sur lequel repose sa formation. J'ai établi une corrélation entre l'induction de NEAT1 et l'activation de l'IRES du FGF1 lors de l'hypoxie. De plus, la déplétion de NEAT1 entraine une inactivation de l'IRES, suggérant que cet ARN non codant est un ITAF. J'ai mis en évidence qu'une autre protéine du paraspeckle, PSPC1, est également un ITAF. Une analyse de la composition de l'IRESome par spectrométrie de masse m'a permis d'identifier trois candidats supplémentaires : hnRNPM, rps2 et Nucléoline. Ayant élargi cette étude aux autres IRES étudiés dans le chapitre 1, nous avons démontré que p54nrb et PSPC1 sont capables d'activer plusieurs IRES alors que l'ARN non codant NEAT1, est un ITAF activateur de tous les IRES testés. NEAT1 paraît donc être la clé de l'activation des IRES, et donne au paraspeckle la fonction nouvelle de plateforme d'assemblage de l'IRESome dans les cardiomyocytes en réponse à l'hypoxie. Dans un troisième chapitre, nous avons élargi l'étude à d'autres types cellulaires, les carcinomes mammaires métastatiques et non-métastatiques 4T1et 67NR. NEAT1 est induit en corrélation avec l'activation de l'IRES du FGF1 lorsque ces cellules sont soumises à l'hypoxie, suggérant que le rôle de NEAT1 et du paraspeckle dans le contrôle de la traduction concerne l'hypoxie tumorale comme l'ischémie. Ainsi, ces travaux de thèse révèlent le grand potentiel de NEAT1 en tant que nouvelle cible thérapeutique
Translation is a highly regulated step of gene expression. During cellular stress, global protein synthesis is blocked but translation of specific subsets of mRNAs is activated by alternative mechanisms. One of these mechanisms involves an RNA structure called IRES (Internal Ribosome Entry Site), that enables the recruitment of the translational machinery in a 5' cap-independent manner. IRES activity is regulated by factors called ITAF (IRES trans-acting factor). Hypoxic stress occurs in different pathologies such as cardiac ischemia and cancer. In response to stress, the cell produces (lymph)angiogenic growth factors that stimulate blood and lymphatic vessel formation, allowing reperfusion of the injured area in ischemic heart, or stimulation of tumor growth and metastatic spread in cancer. My thesis project is focused on identification of ITAF-controlled translation of (lymph)angiogenic growth factor mRNAs during hypoxia. The first part of my thesis addresses translational regulation in hypoxic cardiomyocytes. A semi-global study showed that (lymph)angiogenic genes are mostly regulated at the translational level. Furthermore IRESs of mRNAs coding (lymph)angiogenic growth factors are activated in early hypoxia while IRESs of mRNAs non-related to (lymph)angiogenesis are activated later. Finally, we have identified a new ITAF, vasohibin (VASH1), that specifically activates the FGF1 IRES, but not the other IRES tested. A PCR array study indicates however that VASH1 is able to inhibit or stimulate translation of numerous mRNAs. Identification of a global mechanism of FGF and VEGF IRES activation is the main focus of the second chapter of my thesis. Considering that VASH1 is not a common ITAF, I searched for new candidates. I started from the observation that another ITAF previously identified in the laboratory, P54nrb, is also a component of the paraspeckle, a nuclear body formed during cellular stress. We make the hypothesis that other paraspeckle components could be ITAFs, particularly the backbone of the paraspeckle, the long non-coding RNA NEAT1. I established a correlation between NEAT1 induction in hypoxia and FGF1 IRES activation in cardiomyocytes. Moreover, NEAT1 depletion leads to inactivation of the FGF1 IRES, suggesting that NEAT1 is an ITAF. I also highlighted that another paraspeckle component, PSPC1, has an ITAF function. Analysis of IRESsome composition by mass spectrometry allowed me to identify three other candidates: hnRNPM, Rps2 and nucleolin. We then expanded the study to the other IRESs studied in chapter 1 and demonstrated that P54nrb and PSPC1 are able to activate several IRES whilst the non-coding RNA NEAT1 is a positive ITAF of all tested IRES. Thus NEAT1 seems to be the key of IRES activation, and confers on the paraspeckle the novel function of assembly platform for IRESsome formation in cardiomyocytes during hypoxia. In a third chapter, we investigated the same way in other cellular types, metastatic and non metastatic mammary carcinoma 4T1 and 67NR. NEAT1 is induced in correlation with FGF1 IRES activation when the cells are subjected to hypoxia, suggesting that the role of NEAT1 in translational control can be associated to tumoral hypoxia as well as to ischemia. Thus, this work reveal the unique potential of NEAT1 as a therapeutic target

The 2-oxoglutarate and ferrous ion dependent oxygenases are a super family of enzymes that are involved in a wide range of biological processes regulated trough the mechanism of post-translational modification (PTM). Such biological processes include hypoxia sensing (through regulating HIF transcription), fatty acid metabolism (through carnitine production), transcriptional regulation (through demethylation of histones), and collagen structure formation (through proline and lysine hydroxylation). To understand the underlying mechanisms of such regulatory processes, and to develop clinically useful inhibitors, and thereby regulate these processes in living organisms, requires sensitive methods for monitoring enzyme activity. The use of indirect methods such as quantification of reaction products (14CO2 or succinate) can be problematic, as both products can result from competitive reactions. Alternative direct measurement of substrate modifications using mass spectrometry-based proteomics can be applied; however, (1) for this technique the limit of detection is often prohibitive, (2) the method is best suited for the confirmation of known modifications, rather than for the discovery of new modifications, and (3) sequence coverage may often be only 60%, and therefore many modifications can be missed. The aim of the research presented in this thesis was to develop amino acid analysis and to apply these methods to the identification and quantification of PTMs catalysed by 2-oxoglutarate and ferrous ion dependent oxygenases. A range of LC-MS approaches were investigated including: (1) C18 reversed phase chromatography of quinoline derivatised amino acids, (2) ion paring chromatography, and (3) mixed mode chromatography with either UV, or conventional molecular MS, or isotope ratio mass spectrometry detection. Analysis of the elution patterns for those separation techniques enabled estimation of the retention parameters of modified amino acids and the identification of the modifications, where no standards were available. The most sensitive approach developed employed mixed mode chromatography coupled to isotope ratio mass spectrometry which was optimised for the analysis of modified amino acids. This was shown to have a limit of detection two orders of magnitude lower (0.01μM) than other conventional mass spectrometry techniques. Using amino acid labelling in cell culture, a quantification protocol was developed which employed a non-labelled internal standard and selectively labelled cell culture. The method was shown to be suitable for both very accurate quantification at low concentration levels and metabolic studies, allowing us to track back the modifications to their precursors. The analytical methods developed for amino acid analysis were successfully applied to the analysis of modifications resulting from 2-oxoglutarate and Fe dependent oxygenase activity. Stereochemistry of lysyl hydroxylation in the splicing regulatory protein Luk7L2 by JmjD6 as well as of the self hydroxylation of the JmjD6 was identified. The stereochemistry was shown to be different from that of previously reported for the collagen hydroxyline, hydroxylated by the another member of this enzyme family. mbP4H enzyme was shown to catalyse prolyl hydroxylation of taODD resulting in 4R-hydroxyprolyl. Amino acid analysis was used in order to verify the mechanism of the hBBOX catalysed rearrangement of Mildronate. Using the method developed for the analysis of non-derivatised amino acids the screening of potential substrates of hBBOX enzyme was carried out and two new substrates were identified. The isotope ratio mass spectrometry protocol was applied to the study of histones from cell culture; low levels of hydroxylated and methylated amino acids were quantified. The analytical methods described were developed to complement to the well established proteomics techniques. The methods developed enable investigation into the region- and stereo- chemistry of the modified groups within the modified AA residue and has proved to be a powerful tool of exploratory PTM analysis.

This thesis explores roles of 2-oxoglutarate-dependent (2OG) oxygenases as interfaces that modulate steps in the flow of genetic information in cells in response to oxygen availability. Chapter 1 introduces mechanistic, biochemical and physiological aspects of major subfamilies of 2OG oxygenases, and their established regulatory roles in cells. In addition, structural and functional aspects of the ribosome and the translation process are discussed, with a focus on post-translational ribosome modifications. Chapter 2 investigates histone demethylases, which mediate chromatin-dependent regulation of gene expression and provides proof-of-concept for the rational, structure-guided design of small-molecules for selective inhibition of 2OG oxygenases with roles in cancer and inflammatory disease. Chapter 3 suggests regulatory roles for ten-eleven-translocation (TET)- catalysed DNA hydroxylation; calorimetric and thermal analyses reveal a duplex-stabilizing effect of the epigenetic 5-methylcytosine mark that is reversed upon conversion to 5- hydroxymethylcytosine (also termed the ‘sixth’ DNA base), raising the possibility that 2OG oxygenase catalysis might affect transcription via biophysical effects. Chapter 4 investigates fluoride release assays as a technology to enable medicinal chemistry studies on 2OG oxygenases with roles in fat mass regulation and obesity, cancer and inflammation; studies on the ALKBH5 enzyme show that it is a hypoxically upregulated 2OG oxygenase with a substrate preference distinct from previously characterized ALKBH enzymes. Chapter 5 identifies OGFOD1 as a 2OG-dependent ribosomal protein hydroxylase. OGFOD1 catalysis is conserved from yeast to humans. OGFOD1 catalyses formation of trans-3- hydroxy-L-proline in a highly conserved loop of ribosomal protein S23 proximal to the ribosomal decoding centre, possibly to modulate the interactions of eukaryotic ribosomes with tRNA, mRNA and translation factors in an oxygen-dependent manner. OGFOD1 is the functionally most well-conserved protein-modifying 2OG oxygenase; likewise, ribosomal protein S23 hydroxylation is the most well-conserved post-translational ribosome modification in eukaryotes. Some cell lines require OGFOD1 for proliferation, and scaffolds for OGFOD1- selective inhibitors are developed for use as potential antiproliferative agents and probes for cellular function. Chapter 6 shows the development of assays to investigate whether OGFOD1 catalysis affects ribosome assembly and function, including processivity, accuracy of initiation, elongation and termination, in yeast and mammalian cell lines. Chapter 7 concludes that ribosome hydroxylation might present an additional layer of regulatory complexity by which 2OG oxygenases could enable cells to respond to fluctuating oxygen levels.

The translation represents one of the most crucial processes in the cell. That is why it is often targeted by various regulations. Its initiation phase has a particularly important role in regulatory processes. Initiation of translation usually starts by recognition and binding of canonical eukaryotic initiation factor 4E1 (eIF4E1) to the methylguanosine cap present on the 5' end of the majority of eukaryotic mRNA. The family of 4E translation initiation factors contains two more members - eIF4E2 and eIF4E3. Those two proteins can bind cap structure as well which predetermines it to function in the regulation of translation. Protein eIF4E2 is well known for being a translational repressor in development processes and it takes part in specific miRNA-dependent silencing. It was proven to be able to initiate translation in hypoxia which is consistent with its proposed role in hypoxic tumor cells. The biological roles of the protein eIF4E3 are much less understood. This thesis propounds the picture of the overall functions of all discussed translation initiation factors using cell lines with their overexpression or deletion. Experimental data confirmed the role of the eIF4E2 in the regulation of developmental processes. Cell lines with deleted eIF4E2 and eIF4E3 were characterized based on the influence...

Systemic hypoxia frequently occurs in patients with cardiopulmonary diseases. Maintenance of vascular reactivity and endothelial viability is essential to preserving oxygen delivery in these patients. The role of matrix metalloproteinase-2 (MMP-2) and heme oxygenase-2 (HO-2) in the vascular response to hypoxia were investigated. In the first part of the thesis, the role of MMP-2 in regulating systemic arterial contraction after prolonged hypoxia was investigated. MMP-2 inhibition with cyclic peptide CTTHWGFTLC (CTT) reduced phenylephrine (PE)-induced contraction in aortae and mesenteric arteries harvested from rats exposed to hypoxia for 7 d. Responses to PE were reduced in MMP-2-/- mice exposed to hypoxia for 7 d compared to wild-type controls. CTT reduced contraction induced by big endothelin-1 (big ET-1) in aortae harvested from rats exposed to hypoxia. Increased contraction to big ET-1 after hypoxia was observed in wild-type controls, but not MMP-2-/- mice. Rat aortic MMP-2 and MT1-MMP protein levels and MMP activity were increased after 7 d of hypoxia. Rat aortic MMP-2 and MT1-MMP mRNA levels were increased in the deep medial vascular smooth muscle. These results suggest that hypoxic induction of MMP-2 activity potentiates contraction in systemic conduit and resistance arteries through proteolytic activation of big ET-1. The second part of the thesis investigated oxygen regulation of HO-2 protein and whether it plays a role in preserving endothelial cell viability during hypoxia. HO-2, but not HO-1, protein level was maintained during hypoxia in human endothelial cells through enhanced translation of HO-2 transcripts. Inhibition of HO-2 expression increased the production of reactive oxygen species, decreased mitochondrial membrane potential, and enhanced apoptotic cell death and activated caspases during hypoxia, but not during normoxia. These data indicate that HO-2 is translationally regulated and important in maintaining endothelial viability and function during hypoxia. In summary, the thesis demonstrates the importance of MMP-2 and HO-2 in preserving vascular function during prolonged systemic hypoxia. These enzymatic pathways may, therefore, represent novel therapeutic targets that may be exploited to ameliorate the effects of hypoxia in patients with cardiopulmonary disease.