L'adénocarcinome pancréatique est la 4ème cause de décès liés aux cancers dans les pays occidentaux et constitue un véritable défi, tant l'absence de traitement curatif assombrit son pronostic. Les récurrents échecs des thérapies ciblées soulignent la particularité de sa physiopathologie vis-à-vis des autres cancers et la nécessité d'identifier de nouvelles cibles thérapeutiques. La mutation activatrice de l'oncogène KRAS, considérée comme l'événement initiateur de la carcinogenèse pancréatique, est retrouvée dans plus de 90% des cas. L'activation consécutive des voies de signalisation MAPK et PI3K amorce la transformation tumorale et constitue un trait caractéristique du cancer pancréatique. Si la synthèse protéique est altérée dans de nombreux cancers, elle semble jouer un rôle encore plus important dans le cancer du pancréas, puisqu'elle se situe au carrefour de voies fortement activées. Les altérations des facteurs régulant l'initiation sont majoritaires, et ont pour but de détourner la machinerie traductionnelle, à l'avantage de la cellule cancéreuse. Si la cellule cancéreuse présente un niveau de synthèse protéique globalement élevé, l'augmentation sélective de la traduction de certains ARNm semble définir des comportements propres aux différents types de cancers. L'adénocarcinome pancréatique est caractérisé par une perte d'expression du répresseur traductionnel 4E-BP1 dès les stades précoces de la transformation, qui n'est observée dans aucun autre cancer. L'objectif de ces travaux était de mettre en évidence les processus cellulaires affectés par la perte de 4E-BP1, ainsi que leurs conséquences sur le développement du cancer pancréatique. Mes résultats démontrent que si l'absence de 4E-BP1 est favorable à la régénération tissulaire dans le contexte de la pancréatite, elle confère un avantage prolifératif aux cellules cancéreuses pancréatiques exprimant KRAS muté, en favorisant leur réplication. Cette faculté est acquise au travers d'une dérégulation de la synthèse d'effecteurs décisifs pour l'entrée en phase S et l'initiation de la réplication. Ceci suggère que la perte de 4E-BP1 pourrait constituer un mécanisme de résistance à la chimiothérapie en favorisant la réplication des cellules cancéreuses. En effet l'amorçage de nouveaux foyers de réplication pourrait permettre d'échapper à l'incorporation d'analogues toxiques de nucléosides tels que la gemcitabine. L'utilisation d'inhibiteurs de la traduction pourrait ainsi constituer une nouvelle approche thérapeutique, en bloquant la réplication et en potentialisant l'effet de la chimiothérapie. / Pancreatic ductal adenocarcinoma (PDAC) is the 4th cause of cancer-related deaths in western countries with a 5-years overall survival of 5% that has not improved for years. The lack of bona fide curative therapeutics brings major challenges for the development of tailored therapies. Recent clinical failures remind the particular pathophysiology of PDAC compared to other cancers and the need for new strategies to be uncovered. Mutated KRAS is considered to be the initial event in the onset of pancreatic carcinogenesis, and is found in more than 90% of cases. Consequent activation of MAPK and PI3K signaling pathways primes transformation and is particularly significant in PDAC. While protein synthesis is altered in several cancers, it seems to be highly contributive to pancreatic carcinogenesis, as it stands at the junction of hyperactivated pathways. Alterations in initiation factors are the most common, and lead to a "hijack" of the translation machinery to the advantage of the cancer cell. While global translation rates are generally higher in cancer cells, specialized cancer behaviors seem to rely on the specific translation of subsets of mRNAs. PDAC is characterized by a loss of the translational repressor 4E-BP1 that happens early in the transformation process, and which seems specific of this malignancy. Our results demonstrate that while 4E-BP1 deficiency improves tissue regeneration in the context of pancreatitis, this confers proliferative advantage to pancreatic cancer cells expressing mutated KRAS through increased replication. This ability is acquired through enhanced synthesis of effectors involved in S phase entry and replication initiation. Those results suggest that 4E-BP1 loss may serve as a mechanism of resistance to chemotherapy, by promoting cancer cells replication. Priming of new replication foci could be an escape from incorporation of toxic nucleosides such as gemcitabine. The use of translation inhibitors might be a novel therapeutic approach through replication blocking and chemotherapy potentiation.

Eukaryotic initiation factor-4E (eIF4E) controls the rate of cap-dependent translation initiation and is in turn exquisitely regulated by 4E-BPs. 4E-BP2 binds eIF4E with the highest affinity and is implicated in cancer, and metabolic and neurological disorders. Herein we use NMR, ITC and fluorescence to characterize 4E-BP2 structural and binding properties. Isolated 4E-BP2 is intrinsically disordered, but possesses some transient secondary structural propensities. eIF4E, however, is folded but has a disordered N-terminus. The eIF4E:4E-BP2 interaction is tight (Kd = 10-9 nM) and involves 4E-BP2 C-terminal and canonical binding regions, and the disordered eIF4E N-terminus. 4E-BP2 remains largely disordered upon binding to eIF4E. Noteworthy, high affinity interactions are not necessarily mediated by static structures, and 4E-BP2 binding is not the simple “disorder-to-order” transition observed in many interactions involving disordered proteins. This study offers molecular insights into 4E-BP2 functionality, and lays a foundation for development of novel therapies for cancer and neurological disorders.

Eukaryotic initiation factor-4E (eIF4E) controls the rate of cap-dependent translation initiation and is in turn exquisitely regulated by 4E-BPs. 4E-BP2 binds eIF4E with the highest affinity and is implicated in cancer, and metabolic and neurological disorders. Herein we use NMR, ITC and fluorescence to characterize 4E-BP2 structural and binding properties. Isolated 4E-BP2 is intrinsically disordered, but possesses some transient secondary structural propensities. eIF4E, however, is folded but has a disordered N-terminus. The eIF4E:4E-BP2 interaction is tight (Kd = 10-9 nM) and involves 4E-BP2 C-terminal and canonical binding regions, and the disordered eIF4E N-terminus. 4E-BP2 remains largely disordered upon binding to eIF4E. Noteworthy, high affinity interactions are not necessarily mediated by static structures, and 4E-BP2 binding is not the simple “disorder-to-order” transition observed in many interactions involving disordered proteins. This study offers molecular insights into 4E-BP2 functionality, and lays a foundation for development of novel therapies for cancer and neurological disorders.

Chez les mammifères l’initiation de la traduction et, plus particulièrement, la formation du complexe eIF4F, est principalement régulée par la protéine kinase TOR (Target of rapamycin). Cette voie de régulation fait intervenir les protéines 4E-BP (eIF4E-binding proteins) dont l’activité est modulée par la phosphorylation par TOR. Sous leur forme non-phosphorylée, les 4E-BP se lient au facteur d’initiation eIF4E, empêchent son recrutement dans le complexe eIF4F et inhibent ainsi l’initiation de la traduction. Phosphorylées par TOR, les 4E-BP perdent leur affinité pour eIF4E et sont remplacées par eIF4G ce qui active la traduction. La régulation de l’initiation de la traduction par TOR via 4E-BP a été bien décrite dans plusieurs modèles eucaryotes, tels que la levure, les insectes et les mammifères, mais reste encore obscure chez les plantes. Les recherches réalisées au cours de ma thèse ont permis l’identification de deux protéines homologues de 4E-BP chez Arabidopsis. Ces protéines, que nous avons appelées ToRP1 et ToRP2 (TOR Regulatory Proteins), sont caractérisées par la présence d’un motif consensus indispensable pour la liaison à eIF4E, et qui existe chez les protéines 4E-BP des mammifères ainsi que chez eIF4G et eIFiso4G d’Arabidopsis. La protéine ToRP1 est capable d’interagir spécifiquement avec eIF4E, mais aussi avec TOR via son extrémité N-terminale en système double-hybride de levure. ToRP1 et ToRP2 ont également été caractérisées comme étant des cibles directement phosphorylées par TOR chez Arabidopsis. Deux sérines, en position 49 et 89 dans la protéine ToRP1, ont été identifiées comme des sites potentiels de cette phosphorylation. De plus, l’état de phosphorylation de ces sites affecte l’interaction avec eIF4E en système double-hybride de levure. Par ailleurs, des plants d’Arabidopsis déficients en ToRP1 et ToRP2 renforcent la traduction strictement coiffe-dépendante de l’ARNm CYCB1;1, alors que la surexpression de ToRP1 ou de ToRP2 réprime sa traduction. Ces résultats suggèrent donc que les protéines ToRP, identifiées chez Arabidopsis, sont de nouvelles cibles directes de TOR, qui, par leur phosphorylation, régule l’initiation de la traduction coiffe-dépendante. / The target of rapamycin (TOR) is an evolutionarily conserved kinase that is a critical sensor of nutritional and cellular energy and a major regulator of cell growth. TOR controls cap-dependent translation initiation, in particular the assembly of the eIF4F complex, by modulating the activity of eIF4E-binding proteins (4E-BPs). In their unphosphorylated state 4E-BP proteins sequester eIF4E and repress translation. Upon phosphorylation by TOR, 4E-BPs have a low affinity binding to eIF4E and are replaced by eIF4G thus activating translation initiation. 4E-BPs have been discovered in yeast and mammals but remain to be obscure in plants. Here, we identified and characterized two Arabidopsis proteins termed TOR Regulatory Proteins (ToRPs 1 and 2) that display some characteristics of mammalian 4E-BPs. ToRP1 and ToRP2 contain a canonical eIF4E-binding motif (4E-BM) found in mammalian 4E-BPs and Arabidopsis eIF4G and eIFiso4G. ToRP1 interacts with eIF4E, and, surprisingly, the N-terminal HEAT domain of TOR in the yeast two-hybrid system. ToRP1 and ToRP2 are highly phosphorylated at several phosphorylation sites in TOR-dependent manner in planta. Two of these phosphorylation sites have been identified as—S49 and S89—their phosphorylation status modulates ToRP1 binding to eIF4E in the yeast two-hybrid system. In plant protoplasts, ToRP2 can function as translation repressor of mRNAs that are strictly cap-dependent. Our results suggest that ToRPs can specifically bind the Arabidopsis cap-binding proteins (eIF4E/eIFiso4E) and regulate translation initiation under the control of TOR

No description available.

Thesis (M.Sc.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2008/05/29). Includes bibliographical references.

Dissertation--Fachbereich Katholische Theologie--Münster, 1974. / Bibliogr. p. IX-XXIV. Index.

Doct.-thesis--Linguistics--Haryana, India, 1971. / Bibliogr. p. 89-101. Index.

The 52 kDa intermediate filament protein desmin plays an important role in force transmission in skeletal muscle by connecting myofibrils at Z-lines and to the sarcolemma. Desmin content in muscle adapts to contractile activity and may be involved in cellular signaling mechanisms responsible for muscle growth. Purpose: To compare signaling responses of the mTOR pathway in wild type (WT) vs desmin knock out (KO) mice. Methods: WT (n=12) and KO (n=12) mice were exposed to high frequency electric stimulation of the left hindlimb to elicit an acute response of the mTOR pathway. Non-stimulated right hindlimbs were used as a within animal control. Right and left TA and EDL muscles were dissected 30 min post-stimulation and examined for changes in mTOR, 4E-BP1 and p70S6K. Results: Relative to WT control samples, total mTOR and total 4E-BP1 content was higher in KO control samples. Electrical stimulation resulted in an increase p70S6K phosphorylation in WT and KO animals however there was no difference between the groups. 4E-BP1 phosphorylation was increased in WT but not KO following electrical stimulation. There was no change in mTOR phosphorylation in response to stimulation in WT or KO. Conclusion: The absence of desmin in skeletal muscle does not impair the phosphorylation of p70S6K demonstrating that a tensile load on the muscle will likely result in an increase in protein synthesis. Elevated levels of total mTOR and 4E-BP1 may imply an adaptation to increase sensitivity to growth stimuli in the muscle.

A diferenciação das células THP-1 em macrófagos, induzida por PMA, é associada ao aumento da expressão de PPAR. A UTR 5` de PPAR regula negativamente sua síntese, porém, o mecanismo molecular envolvido não foi esclarecido. Neste estudo, o estado traducional das células THP-1 diferenciadas por PMA foi investigado em associação à superprodução de PPAR. A presença de uORFs no transcrito de PPAR, contendo códons de iniciação compatíveis com seqüências de Kosak, poderia ser a causa do efeito inibitório da UTR 5`. A incorporação reduzida de L-[U-14C]leucina revelou que a superprodução de PPAR ocorre durante inibição global da tradução, confirmada pela redução dos polissomos. Além disso, desfosforilação de 4E-BP1 foi observada após tratamento com PMA e é associada a inibição da iniciação da tradução e estimulação da tradução dependente de IRES. De fato, a estrutura da UTR 5` de PPAR apresenta características de transcritos que formam IRES. Assim, a produção de PPAR pode ser regulada por IRES e ocorre concomitantemente com a inibição da tradução dependente de cap / The differentiation of THP-1 cells in macrophages, induced by PMA, is associated to overexpression of PPARb. Previous studies have shown that the PPARb 5\' UTR negatively regulates its expression. In our study the translational status of PMA-differentiated THP-1 cells was investigated in association to PPARb overexpression. Putative compatible Kosak initiation codons were identified in the PPARb uORFs and could be involved in the inhibitory effect of 5\' UTR. Decreased incorporation of L-[U-14C]leucine in proteins revealed that the overproduction of PPARb in PMA-differentiated THP-1 cells coincides with a global decrease in the protein synthesis process. Translation impairment was confirmed by polysome profile assay. An intense dephosphorylation of 4E-BP by PMA treatment was observed. Dephosphorylated 4E-BP causes inhibition of eIF4E cap-dependent translation initiation and favors IRES-dependent translation. The PPARb 5\' UTR structure has some characteristics that resemble the one described for IRES. Therefore, the PPARb production may be controlled by IRES