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Li, Qiyu. "Eukaryotic translation initiation factor eIF4AIII is functionally distinct from eIF4AI and eIF4AII." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0035/MQ64391.pdf.
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Li, Qiyu 1972. "Eukaryotic translation initiation factor eIF4AIII is functionally distinct from eIF4AI AND eIF4AII." Thesis, McGill University, 1999. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=30687.
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eIF4A is an RNA-dependent ATPase and ATP-dependent RNA helicase that is thought to melt the 5' proximal secondary structure of eukaryotic mRNAs to facilitate attachment of the 40S ribosomal subunit. eIF4A functions in a complex with two other initiation factors (eIF4E and eIF4G). Two isoforms of eIF4A, eIF4AI and eIF4AII, are functionally indistinguishable. A third member of the eIF4A family, eIF4AIII, whose human homolog exhibits 65% amino acid identity to human eIF4AI, has also been cloned from Xenopus and tobacco, but its function in translation has not been characterized. In this report, human eIF4AIII is characterized biochemically. While eIF4AIII, like eIF4AI, exhibits RNA-dependent ATPase activity and ATP-dependent RNA helicase activity, it fails to substitute for eIF4AI in an in vitro reconstituted 40S ribosome binding assay. Instead, eIF4AIII inhibits translation in a reticulocyte lysate system. In addition, whereas eIF4AI binds independently to the middle and carboxy-terminal fragments of eIF4G, eIF4AIII binds to the middle fragment only. These functional differences between eIF4AI and eIF4AIII suggest that eIF4AIII plays an inhibitory role in translation under physiological conditions.
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Shahbazian, David. "Eukaryotic initiation factor 4B (eIF4B) : regulation by signaling pathways and its role in translation." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=115902.
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Due to the high energetic expenditure for the cell, the protein biosynthesis in eukaryotes is an extensively controlled process predominantly regulated at the ribosomal biogenesis and translation initiation steps. The ribosomal biogenesis defines the global translational aptitude of the cell. It is a mainly nucleolar process which is regulated at multiple steps (e.g. transcription, rRNA processing and modification, ribosomal protein translation etc). However, the most extensively regulated and the rate limiting step of translation is the initiation. Multiple eukaryotic translation initiation factors (eIFs) function to facilitate this priming step of translation. The initial recognition of the mRNA molecule happens through the 5' cap structure found in all mRNAs of nuclear origin. This event is mediated through the recruitment of heterotrimeric complex eIF4F consisting of cap-binding protein eIF4E, scaffolding protein eIF4G and the RNA helicase eIF4A unwinding secondary structures found in 5'UTR of mRNA and thus thought to facilitate the scanning process. The helicase activity of elF4F complex or of eIF4A alone is further potentiated by eIF4B in vitro. The latter protein is at the focus of present thesis.Signal transduction regulates multiple cellular processes including mitogenesis, differentiation, apoptosis, chemotaxis etc. Signaling pathways also regulate ribosomal biogenesis to coordinate mitogenic cues, nutrient and energy availability with the translational capacity of the cells. Mounting evidence links PI3K-Akt-mTOR and Ras-MAPK cascades to the translational control. In this thesis, I show that PI3K/mTOR and MAP kinase cascades converge to phosphorylate eIF4B on Ser422. This phosphorylation results in an increased interaction with eIF3, an essential factor bridging between eIF4F and the small ribosomal subunit. Physiological significance of eIF4B phosphorylation on Ser422 has been demonstrated by the stimulatory effect of eIF4B Ser422Asp phosphomimetic mutant on cap-dependent translation. Taken together, this represents a new paradigm of translational control mechanism regulated by signaling crosstalk. The function of eIF4B in vitro is well characterized but its in vivoeffects are disputed in literature. To address this I established HeLa cell line stably expressing shRNA targeting eIF4B. eIF4B silencing inhibits proliferation rates and anchorage-independent growth. Expression of luciferase reporter gene containing 5' terminal oligopyrimidine tract (TOP) is selectively repressed in eIF4B-silenced cells and can be rescued by exogenous eIF4B regardless of Ser422 phosphorylation status. Moreover, the de novo synthesis rates of endogenous ribosomal proteins in serum starved cultures recapitulate the luciferase reporter assay data. Utilizing polysomal analysis, I was able to show more significant inhibition of translation initiation in serum starved eIF4B-silenced cells. Our attempt to discover novel eIF4B-interacting proteins by Mass Spectrometry approach led to the identification of nucleolar RNA helicase DDX21. Confocal microscopy has shown partial co-localization of tagged eIF4B and DDX21 in nucleolar periphery. Pulse chase experiments metabolically labeling rRNA show an attenuated 28S rRNA production and concomitant accumulation of 36S intermediates in eIF4B-silenced cells. Since ribosomal biogenesis is highly coordinated process and requires strict stoichiometry maintenance of ribosomal components the observed inhibition of rRNA processing could be consequential to the decreased ribosomal protein expression. However, given the fact that eIF4B is associated with the nucleolar pre-ribosomal particle complexes its direct effect on rRNA processing cannot be ruled out. Regulation of ribosomal biogenesis by translation initiation factor may represent an important control mechanism allowing cells to co-ordinate these two processes.
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Combe, Jonathan P. "The isolation and functional analysis of the eukaryotic translation initiation factor eIF4E from tobacco." Thesis, University of Leicester, 1998. http://hdl.handle.net/2381/29777.
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An investigation into the regulatory characteristics of plant eIF4E was initiated. Two cDNAs, NeIF4E1 and NeIF4E2 encoding putative tobacco eIF4E homologues were isolated from a pollen cDNA library. NeIF4E2 contained an incomplete open reading frame (ORF) truncated at the 5' terminus, whereas NeIF4E2 consisted of a complete ORF, encoding a predicted 222 amino acid polypeptide. The predicted polypeptide sequences of NeIF4E1 and NeIF4E2 were 95% identical. The complete NeIF4E1 translation product was 67, 69 and 64% identical to wheat, rice and the Arabidopsis eIF4E, respectively. The expression pattern of NeIF4E in tobacco was investigated at the mRNA and protein level. The latter involved the raising of rabbit polyclonal antibodies against bacterially expressed NeIF4E1 protein. Both NeIF4E1 mRNA and protein were detected in all tissues and developmental stages investigated. To investigate whether NeIF4E was essential for tobacco translation, NeIF4E1 antisense constructs, incorporating either the constitutive CaMV 35S and pollen-specific lat52 promoter, were introduced into tobacco by stable genetic transformation. No visible phenotype was obtained with either construct, although NeIF4E protein was reduced by more than 50% of wild type levels with the CaMV35S-NeIF4E1-antisense transgene. The effect of overexpressing NeIF4E1 on plant gene expression was also investigated. Sense NeIF4E1 constructs containing the CaMV35S and lat52 promoters were transiently expressed, with a luciferase test plasmid, in leaves and pollen respectively. Luciferase gene expression was consistently inhibited by NeIF4E1 overexpression. Further experiments with mutant NeIF4E derivatives indicated the inhibition was exerted by the NeIF4E polypeptide not the transcript.
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Ruud, Kelley Astrid. "Identification and characterization of a novel cap-binding protein from Arabidopsis thaliana and of the wheat eukaryotic initiation factor eIF4G /." Digital version accessible at:, 1999. http://wwwlib.umi.com/cr/utexas/main.
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Jones, Grant D. "Discovery, Phylogenetic Analysis, and Functional Characterization of a Unique Family of Eukaryotic Translation Initiation Factor 4E, eIF4E, From Amphidinium carterae, a Marine Dinoflagellate." Thesis, University of Maryland, Baltimore, 2016. http://pqdtopen.proquest.com/#viewpdf?dispub=10118645.
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This study investigates the eIF4E family members in Dinoflagellates. Dinoflagellates are eukaryotic algae with large genomes and a minimal role for transcriptional regulation. All mRNA in dinoflagellates is trans -spliced with a 22-nucleotide 5'-spliced-leader sequence bearing a multi-methylated cap. Like other eukaryotes, dinoflagellates encode multiple eIF4E family members that are anticipated to fulfill a range of functions. Three distinct and novel clades of eIF4E have been recognized in dinoflagellates that are separate from the three metazoan classes of eIF4E. The dinoflagellate Amphidinium carterae encodes eight eIF4E family members while Karlodinium veneficum encodes fifteen eIF4E family members. I assayed six of these family members from A. carterae for expression levels, m7GTP binding, yeast knockout complementation and affinity for three mRNA cap analogs using surface plasmon resonance (SPR). Transcripts of each are expressed through a diel cycle, but only eIF4E-1 family members and eIF4E-2a are expressed at the level of protein. Recombinant eIF4E-1 family members and eIF4E-3a, but not eIF4E-2a, are able to bind to m 7GTP-agarose beads. Of the clade 1 eIF4Es, only eIF4E-1a and -1d1 complement a S. cerevisiae strain conditionally deficient in functional eIF4E, consistent with their function as translation initiation factors. However, only eIF4E-1a can be recovered from A. carterae extracts by m7GTP affinity binding. Using SPR analysis, the affinity of A. carterae eIF4E-1a for m7GTP is lower than that of murine eIF4E-1A. By the same analysis, A. carterae eIF4E-1a has a higher affinity for m7GpppG than m7GTP. In addition, K. veneficum eIF4E-1a1 displays many of the same characteristics as A. carterae eIF4E-1a. Four eIF4E-1 and one eIF4E-2 family members from K. veneficum were characterized for m7GTP binding capacity, only the eIF4E-1 family members can be pulled down with m7GTP. Three eIF4E family members were tested for their ability to interact with a putative eIF4E-interacting protein, although none interacted. Overall, the eIF4E-1a sub-clade emerges with characteristics consistent with the role of a prototypical translation initiation factor. These initial analyses will allow for a better understanding of specific translational control of gene expression through mRNA recruitment in the unique dinoflagellate lineage.
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Metz, Anneke Maria. "Function of the wheat eukaryotic initiation factors eIF(ISO)4G and eIF4B in translation /." Digital version accessible at:, 1998. http://wwwlib.umi.com/cr/utexas/main.
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Green, Simon Richard. "Molecular analysis of eukaryotic initiation factor 2#alpha#." Thesis, University of Kent, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.330170.
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Bottari, Nicolas. "Putative role of eukaryotic initiation factor 4AIII in the nucleus." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=78325.
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Nonsense-mediated decay (NMD) is a cellular event by which messenger RNAs (mRNA) possessing a premature termination codon (PTC) are rapidly degraded to avoid production of incomplete proteins. NMD is a translation-dependent cytoplasmic and nucleus-associated event. Eukaryotic initiation factor 4AIII (eIF4AIII) is a RNA-helicase protein found mostly in the nucleus. It is related to the eIF4AI and eIF4AII proteins, which are localized to the cytoplasm where they are involved in translation initiation. The role of eIF4AIII in nucleus-associated NMD was investigated in this work. HeLa cells were treated with small interfering RNA (siRNA) targeted against eIF4AIII. Immunoblot analysis revealed 45 to 93% knockdown of eIF4AM protein levels compared to nontransfected cells. RNA from siRNA-treated HeLa cells stably transfected with the T-cell receptor-beta (TCR-beta) gene possessing a PTC was analyzed by Northern blotting. TCR-beta mRNA levels were reduced in nontransfected cells, increased in eIF4AIII knockdown cells, and reduced in exogenous HA-eIF4AIII rescued cells. A helicase-dead mutant of eIF4AIII (PRRVAA) also rescues the TCR-beta mRNA decay. Pulse-labelling experiments using radioactive 35S-methionine revealed that eIF4AIII knockdown cells had a reduced (~50%) translational rate compared to nontransfected cells. This data suggests that eIF4AIII is involved in nucleus-associated nonsense-mediated decay and also has a role in general translation.
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Fennell, Clare. "Characterisation of the eukaryotic initiation factor 2alpha kinases of Plasmodium falciparum." Thesis, University of Glasgow, 2008. http://theses.gla.ac.uk/527/.
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Malaria remains a devastating disease with respect to both mortality and the constraints it places on the economic development of the countries in which it is endemic. Our laboratory is seeking new antimalarial targets, by characterising the protein kinases of the most lethal human malaria parasite, Plasmodium falciparum. As central components of many diverse signalling pathways, protein kinases are crucial for the control of proliferation and differentiation in other eukaryotes; we hypothesise that they play similar roles in P. falciparum. The life cycle of P. falciparum is complex, consisting of a series of tightly controlled stages of division and differentiation. In the related apicomplexan parasite Toxoplasma gondii, stress stimuli have been implicated in an important differentiation step, from rapidly dividing tachyzoites, to quiescent bradyzoites (which enable immune evasion). Evidence suggests that stress may also contribute to an essential differentiation stage, gametocytogenesis, in P. falciparum. In yeast and metazoans, part of the stress response is mediated through phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha), which results in selective translation of mRNAs encoding stress response proteins. Post-transcriptional control of gene expression is suspected to play an important role in P. falciparum. Importantly, the Goldberg laboratory recently demonstrated that similarly, in P. falciparum the eIF2alpha orthologue is phosphorylated in response to starvation. Here we identify the P. falciparum orthologue of the translation initiation factor eIF2alpha and provide bioinformatic evidence for the presence of three eIF2alpha kinases in P. falciparum; PfeIK1, PfeIK2 and PfPK4, only one of which (PfPK4) has been described previously (Mohrle et al., 1997). We show that one of the novel eIF2alpha kinases, PfeIK1, is able to phosphorylate P. falciparum eIF2alpha in vitro. In addition, initial experiments support previous observations that PfPK4 is indeed an active protein kinase (Mohrle et al., 1997). We present evidence that PfPK4 is essential for asexual growth, which precludes straightforward reverse genetics studies aiming to determine its possible role in gametocytogenesis. In contrast, transgenic parasites allowed us to show that neither PfeIK1 nor PfeIK2 are required for asexual growth, or sexual development of the parasite in the mosquito vector. However, preliminary evidence (requiring confirmation) may indicate that parasites lacking PfeIK1 over-express PfPK4, which would suggest that PfeIK1 may play an important function in the parasite. This study strongly suggests that a mechanism for versatile regulation of translation by several kinases with a similar catalytic domain, but distinct regulatory domains, is conserved in P. falciparum.
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Andrieu, Claudia. "Etude des mécanismes d'action d'Hsp 27 responsables de l'évolution androgéno-indépendante des cancers de la prostate : mise en évidence de nouvelles stratégies thérapeutiques." Thesis, Aix-Marseille, 2012. http://www.theses.fr/2012AIXM4007.
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Le cancer de la prostate (CaP) est devenu un véritable problème de santé publique dans les pays industrialisés. L'hormonothérapie reste le traitement de première ligne le plus efficace dans les cancers avancés mais il n'empêche pas la progression vers un stade androgéno-indépendant (AI), pour lequel la chimiothérapie s'avère peu efficace. Une des stratégies pour améliorer les thérapies actuelles consiste à cibler des gènes de survie surexprimés dans les CaPs AI afin de restaurer la sensibilité aux traitements du CaPs. Hsp27, protéine surexprimée dans ces cancers, à un effet cytoprotecteur qui engendre une résistance aux traitements. Elle est maintenant reconnue comme une cible thérapeutique importante. Rocchi et al. ont développé un oligonucléotide antisense (ASO) de deuxième génération (OGX-427) qui cible l'ARNm d'Hsp27. OGX-427 est actuellement en essai clinique phase II chez des patients atteints de CaPs au Canada et aux Etats-Unis. Mon projet de thèse a porté sur l'étude des mécanismes d'action d'Hsp27 impliqués dans l'évolution AI du CaP. Cette étude a pour but d'améliorer la sureté pharmacologique d'OGX-427, mais aussi d'identifier de nouvelles cibles thérapeutiques visant spécifiquement les cellules tumorales. Mes travaux de thèse ont montré que lors d'un stress cellulaire induit par hormonothérapie et/ou chimiothérapie, Hsp27 interagit avec le facteur eucaryotique d'initiation de la traduction eIF4E et le protège de sa dégradation par la voie ubiquitine/protéasome. Ceci maintient la synthèse protéique et engendre une survie cellulaire impliquée en partie dans l'effet cytoprotecteur médié par Hsp27Prostate cancer (PC) has become a real public health issue in industrialized countries, mainly due to patients' relapse by castration-resistant (CR) disease after androgen ablation. One strategy to improve current therapies in advanced PC involves targeting genes that are activated by androgen withdrawal, either to delay or prevent the emergence of the CR phenotype. Hsp27 is over-expressed in this cancer and has been shown to play a cytoprotective role leading to treatments resistance. This protein is now considered as promising therapeutic target. Rocchi, P. et al. developed and patented a second generation antisens oligonucleotides (ASO) targeting Hsp27 that has been licensed (OGX-427) and phase II clinical trials are currently in process in PC in Canada and USA. My PhD project focused on the study of Hsp27 action mechanisms involved in CRPC progression. The present study aims to improve pharmacological safety of OGX-427 and to identify new therapeutic targets specific of CRPC cells. The results of my PhD have shown that during cell stress induced by hormone- and/or chemotherapy, Hsp27 interacts with eukaryotic translation initiation factor eIF4E and protects it from degradation by the ubiquitin/proteasome pathway. This maintains protein synthesis and leads to cell survival, partly involved in the cytoprotection mediated by Hsp27. Our work therefore concerned the characterization of the interaction site between Hsp27 and eIF4E in order to identify potential inhibitors of this interaction that could delay CRPC progression
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Carroll, Matthew 1978. "Characterizing the role of eukaryotic elongation factor 2 and eukaryotic initiation factor 4E binding protein in rapamycin-sensitive signaling in Aplysia." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=80235.
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In Aplysia, serotonin mediates behavioral sensitization partly by increasing the strength of the synapse between sensory and motor neurons, a process known as facilitation. The retention of long term facilitation of sensory-motor neuron synapses requires local translation. Indeed, retention of long term facilitation is blocked by rapamycin, an inhibitor of a specific translational pathway. One rapamycin-sensitive target is S6 kinase. S6 kinase activation and the subsequent increased translation of 5 ' terminal oligopyrimidine (TOP) mRNAs that encode components of the translational machinery has been proposed to be important for retention of long term facilitation (Khan et al., 2001). We have cloned one of these TOP mRNAs, the elongation factor eEF2 and showed that serotonin increased the translation of this mRNA in synaptosomes. The phosphorylation of eEF2 may also be regulated by the rapamycin-sensitive system. eEF2 phosphorylation is mediated by the calcium-sensitive eEF2 kinase and blocks translational elongation. Serotonin application decreased eEF2 phosphorylation in synaptosomes and in isolated neurites and this was blocked by rapamycin. This suggests that serotonin-mediated increases in the translation rate can be independent of the regulation of TOP mRNAs. We propose a mechanism involving eEF2 for increasing synaptic specificity of translational control. Stimulation blocks translation at all synapses through calcium entry and phosphorylation of eEF2. This block can be reversed at specific synapses through activation of the rapamycin-sensitive system and dephosphorylation of eEF2.Phosphorylation of the eukaryotic initiation factor 4E binding protein (4EBP) increases the availability of the mRNA cap-binding eukaryotic initiation factor 4E and is also rapamycin-sensitive. We found that 4EBP phosphorylation at threonine 37/46 was stimulated in a rapamycin-sensitive manner in Aplysia neuronal processes, although it is possible that other 4EBP phosphorylation sites are also rapamycin-sensitive.
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Livingstone, Mark. "A nuclear role for the eukaryotic translation initiation factor 4E-binding proteins." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=97024.
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The regulation of mRNA translation is crucially important in determining which cellular proteins are produced in response to intracellular and extracellular cues. The resulting collection of functional proteins determines which physiological processes a given cell will carry out; therefore, the deregulation of protein production is strongly implicated in diseases, such as cancer, in which cells fail to appropriately respond to stimuli. The mammalian target of rapamycin(mTOR) signaling pathway, which links amino acid, growth factor, and energy availability to mRNA translation resulting in cellular growth and proliferation, is frequently deregulated in cancer and is an active target for drug discovery. Among the effectors of mTOR signaling are the eukaryotic translation initiation factor 4E (eIF4E) binding proteins (4E-BPs), which when phosphorylated bymTOR release the mRNA 5'-cap protein eIF4E to promote translation of progrowth/proliferation mRNAs. While previous biochemical fractionation experiments have suggested that 4E-BP1 is exclusively cytoplasmic, we show using immunoassays that this protein is also present in the nuclei of mammalian cells, where it sequesters eIF4E upon mTOR inhibition. This nuclear eIF4E accumulation is useful as a biomarker for mTOR signaling, as we use it as the read-out for a chemical genetic screen for novel mTOR pathway inhibitors. This discovery of a nuclear eIF4E:4E-BP1 complex opens the door to the potential for4E-BPs to impact nuclear RNA processing events. Evidence for and against iv such a nucleus-specific function for 4E-BPs is evaluated and the nuclear function of 4E-BPs is assessed experimentally.La régulation de la traduction des ARN messagers (ARNm) est d'une importance cruciale afin de contrôler quelles protéines sont produites en réponseaux signaux intra- et extracellulaires. La collection de protéines fonctionnelles quien résulte détermine quels processus physiologiques seront effectués par la cellule. Conséquemment, la dérégulation du contrôle traductionnel est fortement impliquée dans plusieurs pathologies, incluant le cancer, ceci dû au fait que les cellules ne répondent pas de manière appropriée aux stimuli qu'elles reçoivent. Une voie de signalisation impliquée dans la croissance et la prolifération cellulaire qui est souvent dérégulée dans les cancers, la voie de la cible mammifère de la rapamycine (mTOR), et qui intègre la disponibilité en acides aminés, facteurs de croissance et énergie avec la traduction des ARNm, est une cible pharmacologique préférentielle. Parmi les effecteurs de la voie mTOR, on retrouve les protéines s'associant au facteur d'initiation de la traduction eIF4E, les 4E-BP, qui lorsqu'elles sont phosphorylées relâchent la protéine liant la coiffe5' des ARNm, eIF4E, promouvant ainsi la traduction des ARNm encodant des protéines impliquées dans la croissance et la prolifération. En contraste avec les résultats de fractionnement subcellulaires reportés précédemment dans la littérature suggérant que 4E-BP1 est une protéine exclusivement cytoplasmique, nous montrons ici, par essais immunologique, que cette protéine est également v résidente du noyau des cellules mammifères où elle séquestre eIF4E suivant l'inhibition de mTOR. Cette accumulation nucléaire de eIF4E est un biomarqueur de choix que nous avons utilisé comme lecture du niveau d'activité de la voie mTOR lors d'un criblage chimio-génétique entrepris dans le but de trouver de nouveaux inhibiteurs de la voie mTOR. Cette découverte d'un complexe nucléaire eIF4E :4E-BP1 à ouvert la porte à une possible fonction des 4E-BP dans certains processus nucléaires. Les évidences en faveur et en opposition àun tel rôle nucléaire spécifique des 4E-BPs sont évaluées et la fonction nucléaire des 4E-BPs est testée expérimentalement.
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Murphey, Roberta Jean. "Synthesis of deoxyhypusine in eukaryotic initiation factor 4D in rat hepatoma cells." Diss., The University of Arizona, 1989. http://hdl.handle.net/10150/184691.
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The aim of this research was to study the mechanism involved in the synthesis of deoxyhypusine, the intermediate step in the synthesis of the amino acid hypusine. Oeoxyhypusine is derived from the butylamine moiety of a spermidine molecule which is added to the famino group of one lysine in the eukaryotic initiation factor 40 (eIF-4D). Initially, a hepatoma tissue cell (HTC) lysate with a pH of 9.5 in glycine buffer and with a depleted spermidine pool supported deoxyhypusine synthesis in protein. Since CHES buffer was as efficient as glycine buffer, the synthesis of deoxyhypusine was pH dependent (optimum ∼9.2) and not buffer dependent. Next, several inhibitors were used in the cell-free system to block deoxyhypusine synthesis. Only guazatine, a plant amine oxidase inhibitor, completely inhibited deoxyhypusine synthesis. This suggested that an oxidase was involved in deoxyhypusine synthesis. In addition factors were investigated as possible allosteric stimulators of deoxyhypusine formation. NAD⁺, NADH, FAD⁺, FMN⁺, and as nicotinamide were tested for effects on deoxyhypusine formation. NAD⁺ was the most efficient stimulator, but NAOH and nicotinamide also stimulated deoxyhypusine formation. Although these factors increased the synthesis of deoxyhypusine, these assays were done in buffer with low concentrations of spermidine. When the spermidine pool was replenished, these effects were diminished. Thus, it appeared that NAD⁺ may lower the apparent K(m) for spermidine without affecting the V(max) of deoxyhypusine synthesis. The inhibition of deoxyhypusine synthesis by guazatine implied the involvement of a polyamine oxidase. Therefore, the effect of oxygen depletion on deoxyhypusine formation was investigated. The depletion of oxygen reduced the level of deoxyhypusine synthesis to 12% of the control. This activity could be restored to 85% by reoxygenation of the lysate. Thus in support of the suggestion made by the guazatine data, a spermidine oxidase in involved in deoxyhypusine formation. The most significant contribution of this work was the development of a cell free system to study deoxyhypusine. This synthesis required an unusually high pH in vitro and required polyamine depletion (Chapter 2). In addition, synthesis requires a unique spermidine oxidase that is blocked by a guazatine and is conditionally stimulated by NAD⁺ (Chapter 3).
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Quenouille-Lederer, Julie. "Bases génétiques et fonctionnelles de la durabilité des résistances polygéniques au virus Y de la pomme de terre (PVY) chez le piment (Capsicum annuum)." Thesis, Avignon, 2013. http://www.theses.fr/2013AVIG0650/document.
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Les résistances génétiques permettent une lutte efficace contre les maladies des plantes cultivées mais sont limitées par les capacités d’évolution des bioagresseurs ciblés. Chez le piment, le fonds génétique peut améliorer la durabilité de la résistance au PVY conférée par le gène majeur pvr23. L’objectif de ma thèse était de caractériser les facteurs génétiques de l’hôte conditionnant la durabilité du gène majeur en répondant aux questions suivantes : (i) Quels sont leurs actions sur l’évolution des populations virales ? (ii) Correspondent-ils aux QTL (quantitative trait loci) de résistance partielle ? (iii) Sont-ils répandus au sein des ressources génétiques du piment ? Différentes expérimentations incluant des tests de résistances, d’évolution expérimentale et de compétition entre différents variants viraux, ont montré que les facteurs du fonds génétique augmentant la durabilité de pvr23 agissaient en : (i) diminuant la concentration virale dans la plante, (ii) en réduisant les probabilités de mutations du PVY vers le contournement du gène pvr23 et (iii) en ralentissant la sélection des variants viraux contournants. La détection de QTL et la cartographie des facteurs génétiques affectant la fréquence de contournement de pvr23 (QTL de durabilité) a mis en évidence quatre régions du génome du piment qui, par des effets additifs ou épistatiques, expliquent 70% de la variabilité phénotypique observée. La cartographie comparée montre que trois des quatre QTL de durabilité co-localisent avec des QTL affectant la résistance partielle, suggérant que les QTL de résistance partielle ont un effet pléiotropique sur la durabilité d’un gène majeur de résistance. L’étude d’une collection de 20 accessions de piment, porteuses de pvr23 ou pvr24(allèle très proche de pvr23) dans des fonds génétiques variés, a montré que les fonds génétiques favorables à la durabilité de ces allèles de résistance sont fréquents dans les ressources génétiques du piment. Ces résultats mettent en évidence que la durabilité d’un gène majeur de résistance peut-être fortement augmentée lorsqu’il est associé à des facteurs génétiques réduisant la multiplication du pathogène. De plus, la fréquence de contournement du gène majeur s’est révélée être un caractère très héritable (h²=0.87) et la détection de QTL affectant ce caractère est possible. La sélection directe pour de tels QTL est donc envisageable et ouvre de nouvelles perspectives pour préserver la durabilité des gènes majeurs de résistance utilisés en sélection variétaleGenetic resistances provide an efficient control of crop diseases but are limited by pathogen adaptation.In pepper, the durability of the pvr23 allele, conferring resistance to Potato virus Y (PVY), was demonstrated todepend on the plant genetic background. The aim of my PhD thesis was to characterize the host genetic factorsaffecting the durability of the major resistance gene pvr23 and to answer to the following question s: (i) What istheir action on the evolution of the viral population? (ii) Is there identity between the QTLs (quantitative traitloci) controlling the partial resistance and the QTLs affecting the durability of pvr23? (iii) Are these genetic factorswidespread among the genetic resources of pepper? Various experiments including resistance testing,experimental evolution and competition between various PVY variants, enabled to show that the genetic factorsaffecting the durability of pvr23 acted in: (i) decreasing the viral accumulation, (ii) decreasing the probability ofacquisition of resistance breaking (RB) mutations by PVY and (iii) slowing down the selection of RB variants. QTLdetection and mapping of genetic factors affecting the frequency of pvr23 RB showed that four loci actingadditively and in epistatic interactions explained together 70% of the variance of pvr23 breakdown frequency.Comparative mapping between these QTLs and QTLs affecting partial resistance showed that three of the fourQTLs controlling the frequency of pvr23 RB are also involved in quantitative resistance, suggesting that QTLs forquantitative resistance have a pleiotropic effect on the durability of the major resistance gene. Analysis of acollection of 20 pepper accessions, carrying pvr23 or pvr24 (allele closely related to pvr23) in various geneticbackgrounds, showed that genetic backgrounds favorable to the durability of the pvr2-mediated resistance arewidespread in the genetic resources of pepper. These results highlight that the durability of a major resistancegene can be strongly increased when associated with genetic factors decreasing the pathogen multiplication.Moreover, the frequency of a major gene RB is a highly heritable trait and QTLs detection for this trait isachievable. The direct selection for such QTLs opens new prospects to preserve the durability of major resistancegenes used by breeders
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Ali, I. K. "The role of eukaryotic initiation factor 4F in translation initiation by linear scanning and internal ribosome binding." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.595445.
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The experiments described in this dissertation used the rabbit reticulocyte lysate translation assay system to investigate what parts of the eIF4F complex are needed for (a) initiation by the scanning ribosome mechanism, and (b) IRES-dependent initiation. To this end, an affinity chromatography method for depleting reticulocyte lysates of eIF4G was developed, in which translation of all mRNAs was compromised, except (as predicted) translation driven by the HCV and pestivirus IRESes. The activity of the FMDV IRES, and that of the closely related encephalmoyocarditis virus (EMCV), in the depleted system was efficiently rescued by low concentrations of the p100 fragment of eIF4G, or even by just the central domain (p50). Deletions from the N-terminus of p50 did not seriously reduce this activity until they invaded the eIF4A interaction site. Surprisingly, a fragment consisting of amino acids 697-969 of eIF4GI was active in supporting IRES-dependent initiation even though it appears to lack the site of interaction with eIF3. Contrary to all expectations, the translation of capped mRNAs by the scanning ribosome mechanism was also restored by these eIF4G fragments which lack an eIF4E binding site, although high concentrations of p100 were needed than for the rescue of EMCV IRES activity. p100 or p50 could also support capped mRNA translation in normal reticulocyte lysates in which the function of the eIF4F complex had been impaired by addition of either m7GpppG cap analogue, or 4E-binding protein-1 (which removes eIF4E from the eIF4F complex), or FMDV L-greatly influenced by the precise nature of the 5’-terminus, whether uncapped or capped with m7GpppG, GpppG or ApppG end groups. Deletion of just 54 amino acids from the N-terminus of p50 resulted in a severe loss of activity towards scanning-dependent initiation, but had little; effect on the function of the EMCV IRES.
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Kinzy, Terri Goss. "Characterization of GTP and aminoacyl-tRNA binding to eukaryotic initiation factor 2 and elongation factor 1." Case Western Reserve University School of Graduate Studies / OhioLINK, 1991. http://rave.ohiolink.edu/etdc/view?acc_num=case1055276346.
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Abdulkarim, Baroj. "The eukaryotic translation initiation factor 2, a hero turned villain in β cells." Doctoral thesis, Universite Libre de Bruxelles, 2017. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/251713.
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The prevalence of type 2 diabetes is increasing dramatically worldwide. Type 2 diabetes is a major health and socio-economic burden. Genetic predisposition and the obesity epidemic, due to sedentary life style and high caloric food intake, are associated with development of type 2 diabetes. Circulating free fatty acids (FFAs), in particular saturated FFAs, are linked with insulin resistance and β cell dysfunction. Following this background we performed RNA sequencing of human pancreatic islets treated with the saturated FFA palmitate to acquire a global image of the islet response to this insult. We identified several stress pathways induced by palmitate with a major induction of the endoplasmic reticulum (ER) stress response. The ER stress response, in particular the PKR-like ER kinase (PERK) branch, has been shown to be induced by saturated FFA. It leads to increased β cell apoptosis both in fluorescence activated cell sorter (FACS) purified rat β cells and human islets. We further clarified the role of this pathway by studying the involvement of the constitutive repressor of eIF2α phosphorylation (CReP) in a monogenic form of diabetes. CReP is a repressor of eukaryotic translation initiation factor 2α (eIF2α) phosphorylation. A direct target of PERK, eIF2α is involved in translational attenuation and induction of apoptosis. We have shown that CReP loss-of-function leads to a new syndrome of young onset diabetes, intellectual disability and microcephaly. The identified R658C mutation abrogated CReP activity leading to increased eIF2α phosphorylation and β cell apoptosis. To further demonstrate the importance of eIF2α dysregulation in β cell demise, we used guanabenz, a chemical inhibitor of growth arrest DNA damage inducible 34 (GADD34). GADD34 is an ER stress-induced repressor of eIF2α phosphorylation. Guanabenz potentiated FFA-mediated ER stress and apoptosis in clonal and primary rat β cells and in human islets through the activation of CCAAT/enhancer binding protein homologous protein (CHOP), downstream of eIF2α. Guanabenz administration in mice impaired glucose tolerance and led to β cell dysfunction. In ex vivo experiments guanabenz also induced β cell dysfunction in mouse and rat islets.In conclusion our data demonstrate that the dysregulation of signaling in the PERK/eIF2α pathway is crucial for β cell demise. Together with previously reported monogenic diabetes caused by loss-of-function mutations in PERK in man and the eIF2αS51A mutation in mice, our findings suggest that a narrow regulation of PERK/eIF2α signaling is central for proper β cell function and survival.Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)
info:eu-repo/semantics/nonPublished
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Hoose, Alex. "Cyclic peptide inhibitors of the eukaryotic translation initiation factor 4E and 4G interaction." Thesis, University of Southampton, 2016. https://eprints.soton.ac.uk/403847/.
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Cancerous tumors require a range of oncogenic proteins to promote cellular proliferation and inhibit apoptotic signals. The effective cap-dependent translation of oncogenic mRNA displays an absolute requirement for certain eukaryotic translation initiation factors (eIF). In particular the interaction of eukaryotic translation initiation factor 4E (eIF4E) with eukaryotic initiation factor 4G (eIF4G) is absolutely required to maintain the malignant phenotype. By contrast the eIF4E / eIF4G interaction is not required for the cap-independent translation of mRNAs that encode homeostatic proteins within healthy cells. As such, the inhibition of cap-dependent translation via disruption of the eIF4E / eIF4G interaction has become an attractive strategy towards the development of novel cancer drugs. A reverse two-hybrid system was constructed with binding fragments of eIF4E and eIF4G within Escherichia coli, in order to mimic the oncogenic PPI found within malignant tissue. Split intein circularization of peptides and protein technology was then utilized to screen a library of DNA encoded cyclic peptides against the eIF4E / eIF4G interaction. Screening isolated four potential cyclic peptide inhibitors of the oncogenic protein-protein interaction that disrupted the eIF4E / eIF4G interaction in Escherichia coli. Putative cyclic peptide inhibitors of the eIF4E / eIF4G interaction were subsequently synthesized by 9-fluorenylmethoxycarbonyl solid phase peptide synthesis in preparation for testing against immortalized cancerous cell lines. A series of assays were devised to determine the effect of compound treatment on translation. Screening of cyclic peptides by MTT cell viability assay determined that two putative inhibitors of the eIF4E / eIF4G interaction elicited a dose-dependent response in MCF7 cells. Further testing of cyclic peptides by a luciferase reporter assay suggested that cyclic peptide treatment had little effect on cap-dependent or cap-independent translation in HeLa cells. This finding was confirmed by 35S labelled methioinine/ cysteine incorporation, which suggested that cyclic peptide treatment had little effect on global translation rates in HeLa cells. m7 GTP pull-down experiments subsequently revealed that cyclic peptides did not disrupt the eIF4E / eIF4G interaction within HeLa cells or within HeLa cell lysate. It is therefore probable that SICLOPPS screening failed to isolate cyclic peptide inhibitors of this oncogenic PPI.
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Gentz, Petra Monika. "Towards understanding the mechanism of dimerisation of Saccharomyces cerevisiae eukaryotic translation initiation factor 5A." Thesis, Rhodes University, 2008. http://eprints.ru.ac.za/1161/.
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Fleming, Keiran Paul. "Liquid crystal NMR : application to a bacterial adhesin and a eukaryotic translation initiation factor." Thesis, Imperial College London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.409641.
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Lazaris-Karatzas, Anthoula. "Eukaryotic translation initiation factor 4E : its characterization as a proto-oncogene and mechanism of action." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=39569.
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Eukaryotic cellular mRNAs have a 5$ sp prime$ cap structure (m7GpppX) that facilitates their binding to ribosomes and is required for efficient translation. An initiation factor, elF-4F, mediates the function of the cap and consists of three subunits. elF-4E, one of the subunits which binds the cap directly, is present in the cell in limiting amounts. We demonstrated that overexpression of elF-4E in NIH 3T3 and Rat 2 fibroblasts causes their tumorigenic transformation. We have also shown that elF-4E cooperates with an immortalizing, nuclear oncogene (ie. v-myc or E1A) to cause transformation of rat embryo fibroblasts. These findings categorize elF-4E as a proto-oncogene. The mode of transformation by elF-4E is similar to that of Ras-like proteins.To further elucidate the mechanism of elF-4E transformation we determined Ras activity in elF-4E transformed REFs. We detected an activation of Ras in eIF-4E overexpressing cells as compared to parental REFs. In addition, overexpression of GAP (GTPase activating-protein), a negative regulator of Ras, suppressed the transformation of REFs by elF-4E.
In summary we have established an important link between Ras, which plays a key role in cellular signal transduction, and elF-4E which is a critical component of the translation machinery.
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Charlton, Jane Laura. "Understanding the biomolecular interactions involved in dimerisation of the Saccharomyces cerevisiae eukaryotic translation initiation factor 5A." Thesis, Rhodes University, 2012. http://hdl.handle.net/10962/d1004118.
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Translation initiation factor 5A (IF5A) is an essential, highly conserved protein found within all eukaryotic (eIF5A) and archaeal (aIF5A) cells. The IF5A protein is unique in that it contains the amino acid hypusine; a two-step post translational modification of a single, conserved lysine residue. Although hypusination of eIF5A is vital for eukaryotic cell viability, the primary role of the protein and its hypusine side chain remain a mystery. eIF5A, initially identified as a translation initiation factor, is not required for global protein synthesis leading to the prevailing proposal that eIF5A is purely involved in the translation of a select subset of mRNAs. Recently a number of mutational studies have focused on the conserved, hypusine-containing loop region of eIF5A where specific residues have been found to be essential for activity without affecting hypusination. It has been postulated that eIF5A exists as a dimer (40 kDa) under native conditions and that these residues may be at the interface of dimerisation. The aim of this research was therefore to conduct a mutational analysis of the loop region in support of this hypothesis. A functional analysis of the Saccharomyces cerevisiae eIF5A mutant proteins K48D, G50A, H52A and K56A revealed that these substitutions impaired growth to varying degrees in vivo with G50A and K48D mutant proteins displaying the most convincing defects. Gel filtration profiles gave unexpected results determining eIF5A mutant and wild type proteins to have a native molecular weight of 30 to 31 kDa, suggesting that the eIF5A oligomeric state may be transitory and subject to certain conditions. The inconclusive results obtained from using gel filtration studies led to an investigation into the feasibility of producing native, hypusinated peptides for future structural studies using nuclear magnetic resonance. Hypusinated and unhypusinated eIF5A were successfully separated into their domains making this a possibility. Finally, this study proposes a role for eIF5A in eukaryotic IRES-driven translation initiation.
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Kazemi, Shirin. "The role of eukaryotic initiation factor 2 alpha phosphorylation pathway in translational control and virus-mediated oncongenesis /." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103165.
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Two important steps of translation initiation include the recognition of the mRNA cap structure by eIF4E and the recycling of erF2. Each step is thought to be regulated independently through the interaction of eIF4E with 4E-BPs and the phosphorylation of the a subunit of eIF2 at serine 51. Phosphorylation of eIF2alpha by dsRNA-dependent protein kinase PKR inhibits protein synthesis in cells subjected to virus infection; therefore, most viruses have evolved mechanism to overcome the deleterious effects of PKR. The human papillomavirus (HPV) E6 oncoprotein contributes to virus-induced pathogenicity through multiple mechanisms including the inhibition of apoptosis and the blockade of interferon action. This study demonstrates a novel function of PKR providing a link between the two mechanisms of regulation of translation initiation. Activation of PKR induces the PI3K-PKB/Akt and FRAP/mTOR pathways leading to S6 and 4E-BP1 phosphorylation upon stress conditions and in response to growth stimuli. Induction of the PI3K pathway antagonizes the apoptotic effects of PKR activation, but does not intervene with its translational inhibitory activity. Investigating functional interaction of HPV E6 and PKR, we determined that BPV-18 E6 protein synthesis is regulated by eIF2alpha phosphorylation. On the other hand, E6 oncoprotein is able to rescue cells from PKR-mediated inhibition of protein synthesis and induction of apoptosis by promoting eIF2alpha dephosphorylation through physical association with GADD34/PP1 holophosphatase complex. These findings demonstrate, for the first time, the ability of PKR to activate a growth-stimulatory pathway; PI3K. Furthermore, it demonstrates role of oncogenic E6 in antagonizing signaling pathways induced by PKR including eIF2alpha phosphorylation and PI3K pathway.
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Zyryanova, Alisa. "A molecule-inhibitor of the integrated stress response regulates activity of mammalian eukaryotic translation initiation factor 2B." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/284208.
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The Integrated Stress Response (ISR) is a conserved eukaryotic translational and transcriptional program implicated in mammalian metabolism, memory and immunity. Although mainly considered to be a protective mechanism, prolonged and severe ISR can result in cell death. The ISR is activated by diverse stress pathways converged on phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2) that inhibits the guanine nucleotide exchange activity of its partner eIF2B and attenuates overall rates of protein synthesis. Numerous mutations in eIF2B are linked to a fatal neurodegenerative disease of vanishing white matter. A new chemical inhibitor of the ISR (ISRIB), a bis-O-arylglycolamide, can reverse the attenuation of mRNA translation by phosphorylated eIF2 protecting mice from prion-induced neurodegeneration and traumatic brain injury. The work presented in this dissertation describes identification of mammalian eIF2B as a cellular target of ISRIB by implementing biochemical, biophysical, structural and chemogenetic methods. The herein reported cryo-electron microscopy-based structure of eIF2B uncovers a novel allosteric site on the translation factor capturing the ISRIB-binding pocket at the interface between its β and δ regulatory subunits. The extensive CRISPR/ Cas9-based screen for ISRIB-resistant and analogue-sensitive phenotypes revealed residues on the eIF2B dimer interface important for ISRIB binding. Based on the results reported in this dissertation along with the similar findings of others the potential molecular basis of ISRIB action, and its implication for the regulation of eIF2B's activity is broadly discussed. The identification of the ISRIB binding pocket away from the known interaction sites between eIF2B and eIF2 is also put into the context of a possible molecular mechanism of eIF2B's guanine exchange inhibition by phosphorylated eIF2. The work described in this dissertation provides new insight into the translational regulation and points to the importance of fine-tuning the activity of translation factors by small chemical molecules.
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Sun, Yingjie. "Single-molecule studies of the eukaryotic translation initiation factor 4A: helicase activity, conformational dynamics and function regulation." Thesis, Boston University, 2013. https://hdl.handle.net/2144/11060.
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Thesis (Ph.D.)--Boston UniversityThe PI3K/Akt/mTOR pathway regulates several cellular functions, including cellular proliferation, growth, and survival. The PI3K/Akt/mTOR pathway converges on the eukaryotic translation initiation factor 4F (eIF4F), making it an attractive molecular target for anti-cancer therapies. As a subunit of eIF4F, eIF4A is known to facilitate binding and scanning of the ribosome by unwinding secondary structures in the 5' untranslated region (UTR) of mRNAs. However, the molecular mechanisms of eIF4A activity have remained elusive. Single-molecule Fluorescence Resonance Energy Transfer (sm-FRET) can probe structural changes and interactions of biological systems in real time, which cannot be observed using bulk techniques. First we directly observe and quantify the helicase activity of eIF4A in the presence of the ancillary RNA-binding factor eIF4H using sm-FRET. We show that eIF4H can significantly enhance the helicase activity of eIF4A by strongly binding both to loop structures within the RNA substrate as well as to eIF4A. Electrophoretic mobility shift assay (EMSA) shows that eIF4H binds to the amino-terminal domain (NTD) but not to the carboxyterminal domain (CTD) of eIF4A. In the presence of ATP, the eIF4A/eIF4H complex exhibits rapid and repetitive cycles of unwinding and re-annealing. ATP titration assays suggest that this process consumes a single ATP molecule per cycle. Second, we directly probe the conformational dynamics of eIF4A, in real time, using smFRET. We demonstrate that the eIF4A in the presence of eIF4H can repetitively unwind the RNA hairpin substrate by transitioning between an "open" and a "closed" conformation using the energy from ATP hydrolysis. Upon binding of an RNA hairpin and ATP, which is mediated by eIF4H, eIF4A adopts a closed conformation; after ATP hydrolysis, eIF4A returns to the open conformation and the RNA duplex is completely unwound. Then the eIF4A releases the RNA and the hairpin is quickly reformed. Third, we find that RNA aptamer and the small molecule hippuristanol can inhibit the binding to the RNA substrate or the helicase activity of the eIF4A/eIF4H complex respectively. The RNA aptamer can directly compete with an RNA hairpin for binding to both eIF4A and eIF4H. Hippuristanol inhibits helicase activity by blocking the conformational change of eIF4A.
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Avison, Matthew B. "The role of phosphorylation in the interaction between eukaryotic initiation factor elF4E and 4E-BP1 in rat adipocytes." Thesis, University of Bristol, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263829.
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Elsby, Rachel Jane. "The Alpha Subunit of Eukaryotic Initiation Factor 2B Is Requisite for EIF2-Mediated Transitional Suppression of Vesicular Stomatitis Virus." Scholarly Repository, 2008. http://scholarlyrepository.miami.edu/oa_dissertations/33.
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Eukaryotic initiation factor 2B (eIF2B) is a heteropentameric guanine nucleotide exchange factor (GEF) that converts inactive eIF2 GDP-bound binary complexes into active eIF2 GTP-bound complexes that can bind initiator t-RNA molecules and ribosomes to begin translation. eIF2B is functionally divided into two subcomplexes: the catalytic core comprised of eIF2B epsilon and eIF2B gamma, and the regulatory core comprised of eIF2B alpha, eIF2B beta and eIF2B delta. While the catalytic subunits are responsible for exerting GEF activity, the regulatory subunits recognize eIF2 and respond to eIF2 alpha phosphorylation. Cellular stress, such as virus infection, inhibits host protein synthesis by activating specific kinases that are capable of phosphorylating the alpha subunit of eIF2, which can then sequester eIF2B to stall guanine nucleotide exchange by a currently unresolved mechanism. Importantly, we demonstrate that loss of eIF2B alpha or expression of a variant of the human eIF2B alpha subunit harboring a single point mutation (T41A) is sufficient to neutralize the consequences of eIF2 alpha phosphorylation, and render primary MEFs significantly more susceptible to vesicular stomatitis virus infection. To extend this analysis, we further exhibit the vital function of eIF2B alpha in protein synthesis through phenotypic studies in yeast. Here, we report that this subunit can sufficiently substitute for its yeast counterpart, GCN3, and reproduce similar growth phenotypes under normal and amino acid deprived conditions. In addition, the human eIF2B alpha-T41A variant was unable derepress GCN4 translation in response to an inhibitor of amino acid biosynthesis in yeast, an activity that requires sensitivity to phosphorylation of the yeast eIF2 alpha homolog, SUI2. Previously, we have demonstrated that vesicular stomatitis virus can infect and replicate to high levels in tumor cells. Moreover, these cells appear to contain defects in eIF2 alpha-mediated translational control, plausibly due to disregulation of eIF2B activity, which overcomes the inhibitory effects of eIF2 alpha phosphorylation. Our data suggest a role for eIF2B, specifically eIF2B alpha, in suppression of translation following virus infection, and imply that this complex may contribute to oncogenic transformation. These results emphasize the importance of eIF2B alpha in mediating eIF2 kinase translation inhibitory activity and may provide insight into the complex nature of viral oncolysis and cellular transformation.
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Long, Ezhou. "Proliferation and apoptosis of bovine mammary epithelial cells : roles of eukaryotic translation initiation factor 4E and Escherichia coli mastitis." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37766.
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Milk yield is dependent on both the number and secretory activity of mammary alveolar cells. The number of cells is controlled by their proliferation and death. In the first study, the effect of eukaryotic translation initiation factor 4E (eIF-4E) on the growth of a bovine mammary epithelial cell line, MAC-T, was investigated. Compared to the parental controls, overexpression of mouse wild-type (wt) eIF-4E in 11A, a subclone of MAC-T cells, increased the growth rate and saturation density (number of cells per well at confluence), whereas overexpression of a mutant eIF-4E (W56A) decreased growth rates and saturation densities. Furthermore, cyclin D1 expression among the 4E-overexpressing and parental cells was compared. Compared to the controls, the amounts of cyclin D1 mRNA and proteins were higher in the cells overexpressing wt eIF-4E but lower in the cells with mutant eIF-4E expression. Our results suggest that altered expression of eIF-4E leads to changes in cyclin D1 expression, which consequently modulate the growth properties of MAC-T 11A cells.Because of its unknown sequence, bovine eIF-4E cDNA was then cloned in the second study. Its coding region consists of 651 nucleotides which encode 217 amino acids (AAs). Bovine eIF-4E cDNA shares 94%, 89% and 94% homology with those of human, mouse and rabbit, respectively. Differences in protein sequences between bovine and human, mouse and rabbit eIF-4E are 2, 4, and 3 AAs, respectively. Furthermore, expression of eIF-4E in bovine mammary tissues at different physiological periods was investigated by Northern blot analysis, using the cloned cDNA as the probe. eIF-4E was not detectable at prepubertal period and expressed at a very low level at the third estrous cycle. In the lactating mammary tissues, eIF-4E was highly expressed. Differential expression of eIF-4E in bovine mammary gland at distinct physiological stages indicates its potential involvement in mammary development.
Cell proliferation and apoptosis were also studied in the Escherichia coli (E. coli)-infected bovine mammary glands in the last study. Both proliferation and apoptosis increased in the mastitic tissue, as determined by immunohistological assays. Compared to the controls, expression of the pro-apoptotic proteins, Bax and interleukin-1beta converting enzyme (ICE), increased at 24 h and 72 h post-infection, whereas expression of the anti-apoptotic protein Bcl-2 decreased only at 24 h post-infusion. Induction of extracellular matrix (ECM)-degrading enzymes, including matrix metal loproteinase-9 (MMP-9), stromelysin-1 (SL-1) and urokinase-type plasminogen activator (uPA), was also observed in the mastitic tissue. Therefore, apoptosis may be mediated through pathways involving the actions of Bcl-2, Bax and ICE, and may partially be accounted by ECM breakdown.
Taken together, our study has demonstrated the effect of eIF-4E on bovine mammary cell proliferation. In addition, its involvement in bovine mammary gland development has been suggested. Finally, increased mammary cell apoptosis and proliferation during E. coli-induced mastitis has been revealed, in association with altered expression of apoptosis-related genes and ECM-degrading enzymes. Understanding the regulation of mammary cell proliferation and death may eventually lead to improvement of milk production.
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Lu, Wei. "The Reciprocal Regulation of Nitric Oxide Synthase and Alpha-subunit of Eukaryotic Initiation Factor 2 Post Ultraviolet B Irradiation." Ohio University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1289006333.
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Talje, Lama. "Structural basis for the recruitment of the SerThr kinase Mnk1 by the scaffolding proteins DAP5 and elF4G." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111548.
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Scaffolding proteins control the localization of protein kinases. During translation, the scaffolding proteins eIF4G and DAP5 recruit the Ser/Thr kinase Mnk1 to phosphorylate the mRNA cap-binding protein eIF4E and modulate translation. Biochemical deletion analysis previously showed that the interaction between Mnk1 and eIF4G/DAP5 is mediated by N-terminal residues in Mnk1 and C-terminal residues in eIF4G/DAP5. Using X-ray crystallography I have determined the structure (1.5 A) of the C-terminal domain of DAP5 (DAP5C). This structure reveals that DAP5C contains two atypical HEAT domains similar to the ones seen previously in the structure of the C-terminal region of eIF4G (4GC). Using ITC I showed that the Kd for the interaction between the N-terminus ofMnk1 and 4GCIDAPSC is 20 muM and 10 muM, respectively. Using NMR chemical shifts we have mapped the residues on both Mnk1 and 4GC/DAP5C which are important for maintaining this interaction. Finally, using SAXS a low resolution configuration of the hMnk1-4GC complex was modeled. It is hoped that an understanding of the structural basis for the recruitment of protein kinases to their sites of action will allow the design of small-molecule compounds that can be used to modulate the location of the kinase and hence its activity.
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Liu, He. "Eukaryotic Initiation Factor 2-associated glycoprotein P67 inhibits the tumorigenicity of Alveolar Rhabdomyosarcoma (ARMS) and involves its differentiation and migration." Kent State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=kent1564585690280704.
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Gatsukovich, Yulia. "Characterization of Eukaryotic Translation Initiation Factor 5A-2 (eIF5A-2) in Arabidopsis thaliana: Effects of Wounding and Pathogen Attack." Thesis, Waterloo, Ont. : University of Waterloo, 2004. http://etd.uwaterloo.ca/etd/ygatsuko2004.pdf.
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Thesis (Ph.D.)--University of Waterloo, 2004."A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Master of Science in Biology." Includes bibliographical references.
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Eshaque, Bithi. "Characterization of Eukaryotic Translation Initiation Factor 5A isoforms (eIF-5A1 & eIF-5A2) using human cell lines as a model system." Thesis, University of Waterloo, 2006. http://hdl.handle.net/10012/1218.
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Eukaryotic translation initiation factor 5A (eIF-5A) is the only known cellular protein that contains the post-translationally derived amino acid, hypusine. Initially, eIF-5A was named as a translation initiation factor because of its capability to stimulate the formation of methionyl-puromycin, which mimics the first peptide bond formation during protein synthesis, under in vitro conditions. Subsequently, however, this proposed function of eIF-5A has been questioned because a similar effect on translation was not observed in situ. Moreover, eIF-5A appears not to be required for general protein synthesis. Rather, there is evidence that it facilitates the translation of specific subsets of mRNAs required for cell proliferation as well as apoptosis. There are two isoforms of eIF-5A in the human genome which have designated eIF-5A1 and eIF-5A2. The objective of the present study was to gain an increased understanding of the roles of eIF-5A1 and eIF-5A2 during apoptosis and cell proliferation using human cell lines as a model system. Apoptosis was induced by treating the cells with Actinomycin D or sodium nitroprusside (SNP), which initiate programmed cell death by different mechanisms. It was observed for both normal and cancer cells that eIF-5A1 protein is up-regulated during apoptosis induced by Actinomycin D or SNP, whereas eIF-5A1 mRNA is constitutively expressed and does not change in abundance during this treatment. The up regulation of eIF-5A1 protein levels in the absence of a corresponding up-regulation in eIF-5A1 mRNA suggests that eIF-5A1 may be post-transcriptionally regulated. Moreover, eIF-5A1 protein up-regulation was stronger in normal cells than in cancer cells. By contrast, eIF-5A2 protein was below detection levels during apoptosis in both normal and cancer cells, although the corresponding transcript was detectable by semi-quantitative RT-PCR. This is attributable to inefficient translation of eIF-5A2 mRNA.
The effects of eIF-5A1 and eIF-5A2 on cell proliferation were examined by modulating the levels of serum in cultures of UACC-1598 cells, which are ovarian cancer cells that express high levels of both isoforms of eIF-5A. Serum starvation, which induces cell cycle arrest and ensuing apoptosis, followed by the re-addition of serum had no effect on the transcript levels of either eIF-5A1 or eIF-5A2. However, eIF-5A1 and eIF-5A2 proteins were both up-regulated within 24 hours of the initiation of serum starvation, and this coincided temporally with the onset of apoptosis as measured by TUNEL and a subsequent decline in viable cells.
The data indicate that eIF-5A1 and eIF-5A2 are both post-transcriptionally regulated and that they have functionally redundant roles in apoptosis.
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Muaddi, Hala. "Phosphorylation of eukaryotic initiation factor 2-alpha at serine 51 is an important determinant of cell survival and adaptation to glucose deficiency." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:8881/R/?func=dbin-jump-full&object_id=92253.
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RUSSO, ARIANNA. "Increasing Eukaryotic Initiation Factor (eIF6) gene dosage stimulates global translation and induces a transcriptional and metabolic rewiring that blocks Programmed Cell Death." Doctoral thesis, Università del Piemonte Orientale, 2018. http://hdl.handle.net/11579/97190.
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Hui, Daniel Jason. "The Mechanism of Protein Synthesis Inhibition by the P56 Family of Viral Stress Inducible Proteins." Connect to text online, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1104848977.
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Hoang, Xuan Chien [Verfasser], and Wilhelm [Akademischer Betreuer] Schäfer. "The role of posttranslational hypusination of the eukaryotic translation initiation factor 5A in Zea mays and Fusarium graminearum / Xuan Chien Hoang ; Betreuer: Wilhelm Schäfer." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2017. http://d-nb.info/1130787044/34.
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Lee, Yun-Young. "Translational regulation of growth arrest and DNA damage-inducible gene GADD34 via its 5' untranslated region upstream open reading frame during eukaryotic initiation factor 2 alpha phosphorylation." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/17442.
Full textAbstract:
Endoplasmic reticulum (ER) stress activates an integrated stress response which causes inhibition of overall protein synthesis via phosphorylation of the eukaryotic initiation factor 2alpha (eIF2alpha). However, ER stress also results in selective translation of mRNAs, one of which is a transcription factor ATF4. ATF4 activates transcription of downstream stress-induced genes such as growth arrest and DNA-damage inducible gene 34 (GADD34) under ER stress. The function of GADD34 is to dephosphorylate eIF2alpha by interacting with protein phosphatase 1, thus leading to recovery of overall protein synthesis and translation of stress-induced transcripts through a negative feedback mechanism. In this thesis, we showed that GADD34 is not only transcriptionally induced, but also translationally regulated for maximal expression under ER stress. Translational regulation of GADD34 was mediated by its 5’ untranslated region (5’ UTR), which was found to contain two upstream open reading frames (uORFs) in human and mouse. It was revealed that the downstream uORF2 is required for basal repression and translational upregulation under ER stress, while the upstream uORF1 is dispensable in this regulation. In addition, the uORF2 is readily recognized and translated, but the uORF1 is bypassed by the scanning ribosomes. Further mutational analysis on the GADD34 5’ UTR demonstrated that the uORF2 and the intercistronic region between the uORF2 and the main ORF are sufficient to direct translation when eIF2alpha is phosphorylated. In this process, the amino acid/nucleotide identity of the uORF2 was not required, but its conserved size was important. The sequence conservation within the intercistronic region also was identified, but changing the length and pyrimidine:purine ratio in this region did not significantly affect translational regulation. Finally, we set up in vitro translation systems where cap-dependent translation is compromised by inhibiting ternary complex and eIF4F formation in order to test GADD34 translational regulation. The results from the current thesis suggest that GADD34 translation is mediated through its 5’ UTR via a unique mechanism, which may serve as a model to understand translational regulation of other uORFs-containing mRNAs under cellular stress.
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Majumdar, Avijit. "Regulation of the activation and activity of the extra-cellular signal regulated kinases 1 & 2 MAP kinase pathway by eukaryotic initiation factor 2 associated glycoprotein p67." [Kent, Ohio] : Kent State University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=kent1209000031.
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Thesis (Ph.D.)--Kent State University, 2008.Title from PDF t.p. (viewed Jan. 26, 2010). Advisor: Bansidhar Datta. Keywords: p67, ERK1, ERK2, oncogenic KRasV12, tumor suppressor. Includes bibliographical references (p. 143-160).
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Gradi, Alessandra. "Translational control in eukaryotes : discovery of a novel human eukaryotic translation initiation factor and its role in the shutoff of host cell protein synthesis following entero- and rhinovirus infections." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=36809.
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The eukaryotic initiation factor (eIF) 4F complex is composed of three polypeptides: eIF4A, eIF4E and eIF4G. eIF4E is the cap-binding subunit; eIF4A is the component which exhibits RNA helicase activity and is thought to unwind the secondary structure present at the 5' leader sequences of mRNAs; eIF4G serves as a scaffold to bring together eIF4E, eIF4A, with eIF3, thus recruiting the mRNA to the 40S small ribosomal subunit. Here, I describe the identification and characterization of a novel homologue of eIF4G that we have termed eIF4GII. We renamed the previously discovered polypeptide, eIF4GI. eIF4GI and eIF4GII are 46% identical at the amino acid level. eIF4GII interacts directly with eIF4E, eIF4A, eIF3 and the poly(A) binding protein. eIF4GII restores cap-dependent translation in a reticulocyte lysate which had been pretreated with rhinovirus 2A protease to cleave endogenous eIF4G. Our findings indicate that eIF4GII is a functional homologue of eIF4GI.In contrast to cellular mRNAs, entero- and rhinovirus RNAs do not possess a cap structure, and their translation is mediated by ribosome binding to an internal ribosome entry site present within the 5' untranslated region. The cleavage of eIF4G induced by the viral protease 2A is thought to be responsible for the shutoff of host protein synthesis in entero- and rhinovirus-infected cells. Nonetheless, in virus-infected cells, a lack of correlation between eIF4GI cleavage and inhibition of host translation is observed. eIF4GII, like eIF4GI, is also cleaved upon viral infection. Here, I present data showing that the kinetics of cleavage of eIF4GII perfectly coincide with the inhibition of cellular protein synthesis observed after entero- and rhinovirus infection. Thus, proteolysis of both eIF4GI and eIF4GII appears to be required for the shutoff of host protein synthesis after entero- and rhinovirus infections.
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Cardin, Eric. "Function of Nck-1 adaptor protein as modulator of elF2alpha phosphorylation by specific elF2alpha kinases and PKR activity." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111905.
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Phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2 (eIF2alpha) on Serine 51 (Ser51) is an early event associated with downregulation of protein synthesis at the level of translation and constitutes a potent mechanism to overcome various stress conditions. In mammals, four eIF2alpha-kinases PERK, PKR, HRI and GCN2, activated following specific stresses, have been involved in this process. Our laboratory has previously demonstrated that the adaptor protein Nck, composed only of Src homology domains and classically implicated in cell signaling by activated plasma membrane receptor tyrosine kinases, modulates translation through its interaction with the beta-subunit of the eukaryotic initiation factor 2 (eIF2beta). Moreover, we reported that Nck-1 overexpression antagonizes the inhibition of translation in endoplasmic reticulum stress condition and prevents the PERK-mediated phosphorylation of the alpha-subunit of eIF2 on Ser51. In this thesis, I demonstrate that the adaptor protein Nck-1 modulates eIF2alpha-kinase-mediated eIF2alphaSer51 phosphorylation in a specific manner. More particularly, I show that Nck-1 overexpression reduces eIF2alpha phosphorylation in conditions activating PKR or HRI as described previously for PERK. In contrast, I observe that overexpression of Nck-1 in mammalian cells fails to attenuate eIF2alphaSer51 phosphorylation in response to amino acid starvation, a stress condition activating GCN2. I further confirm this observation by showing that Nck-1 fails to alter eIF2alphaSer51 phosphorylation in Saccharomyces cerevisiae, for which the sole eIF2alpha-kinase is GCN2. In addition, I report that Nck-1 reduces PKR activation in response to dsRNA. I also find that Nck-1 reduces dsRNA-induced activation of p38 MAPK, a PKR-downstream substrate, and cell death. Finally, I show that Nck-1 interacts exclusively with the inactivated form of PKR in a Src homology domain independent manner. All together these data uncover the existence of a novel mechanism regulating phosphorylation of eIF2alphaSer51 under various stress conditions and identifies Nck-1 as a modulator of the tumor suppressor and antiviral protein kinase PKR.
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Larsson, Ola. "Transcriptome studies of cell-fate and aging /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-296-9/.
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Murphy, Patrick. "Characterisation of critical interactions between translation factors eIF2 and eIF2B." Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/characterisation-of-critical-interactions-between-translation-factors-eif2-and-eif2b(9138d7c8-34b1-4489-8048-a2ac45ef8533).html.
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Eukaryotic translation initiation is a complex and highly regulated process involving the ribosome, mRNA and proteins called eukaryotic initiation factors (eIFs). The overall aim of translation initiation is to position the ribosome at the initiation codon of the mRNA. eIF2, in its GTP-bound conformation, binds the initiator tRNA (Met-tRNAiMet) and delivers it to the 40S ribosomal subunit. When the anticodon of the tRNA is bound to the initiation codon, the GTP on eIF2 is hydrolysed to GDP. The guanine nucleotide exchange factor (GEF) eIF2B regenerates eIF2-GTP. eIF2 and eIF2B are multisubunit/multidomain protein complexes. Because information regarding the interface between each complex is limited, particularly the interface on the eIF2γ subunit, which binds the guanine-nucleotides and Met-tRNAiMet, interactions between the minimal GEF domain of eIF2Bε, εGEF, and eIF2 were mapped using mutagenesis and an in vitro cysteine cross-linking approach, with the cross-linker Mts-Atf-Biotin. Site-directed mutagenesis (SDM) was used to mutate five N-terminal and five C-terminal surface-exposed εGEF residues to cysteines. The mutant alleles were analysed in Saccharomyces cerevisiae and it was found that the gcd6-R574C allele was lethal and the gcd6-T572C was Gcd-. Further gcd6-R574 mutant alleles were also found to be lethal in yeast but expressed in vivo.εGEF-R574C has dramatically reduced GEF activity in vitro and binding assays showed that this mutant has significantly reduced affinity for eIF2. The εGEF-T572C and εGEF-S576C mutants also have severe and minor eIF2-binding defects respectively, while the C-terminal εGEF-Cys mutants have slightly reduced affinity for eIF2. The N-terminal εGEF-Cys mutants cross-link specifically to eIF2γ, while the C-terminal εGEF-Cys mutants interact predominantly with eIF2β. From the data obtained in this study, we propose a new model for eIF2B-mediated guanine-nucleotide exchange that reduces the importance of eIF2β and suggests εGEF resembles other GEFs in binding primarily to its G protein partner eIF2γ.
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Ill-Raga, Gerard. "Study of the pathophysiological role of nitric oxide and nitrative stress in brain: translational effects on the cleavage of the amyloid precursor protein in Alzheimer's disease and post-translational effects on fibrinogen in brain ischemia." Doctoral thesis, Universitat Pompeu Fabra, 2010. http://hdl.handle.net/10803/31907.
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Nitric oxide (NO) is a neurotransmitter involved in memory processes. Currently, the
only recognized physiological signalling pathway controlled by NO is the activation of
guanylyl cyclase. In this thesis, we propose an alternative NO-signalling pathway that
involves the Heme-regulated eukaryotic initiation factor-2a kinase (HRI) and eIF2a
phosphorylation. We have found that the enzyme BACE1, a key protein in Alzheimer’s
disease (AD), is controlled by this novel pathway. This pathway would be involved in
the physiology of memory formation and learning processes. We have also studied how
an external stress factor, the Herpes Simplex Virus 1, can disrupt this cascade leading to
a pathological increase in BACE1 and amyloid ß-peptide (Aß) production. Aß
aggregates forming fibrils that generate free radicals. These react with NO producing
peroxynitrite, which contribute to AD progression. Since NO turns toxic when produced
in a pro-oxidant environment we have also studied the effect of peroxynitrite in Stroke.L’òxid nítric (NO) és un neurotransmissor involucrat en processos de memòria. Actualment, l’única cascada de senyalització fisiològica controlada per NO consisteix en l’activació de la guanilat ciclasa. En aquesta tesi, en proposem una d’alternativa que inclou la fosforilació de eIF2a per la Heme-regulated eukaryotic initiation factor-2a kinase (HRI). Hem mostrat com l’enzim BACE1, una proteïna clau en la malaltia d’Alzheimer (AD), és controlat per aquesta nova cascada de senyalització, que podria estar involucrada en la fisiologia de l’aprenentatge i la memòria. També hem estudiat com un factor d’estrès extern, l’ Herpes Simplex Virus 1, pot pertorbar aquesta cascada donant lloc a increments patològics en BACE1 i pèptid ß-amiloide (Aß). L’Aß agrega formant fibril·les que generen radicals lliures. Aquests reaccionen químicament amb NO produint peroxinitrit, que contribueix a la progressió de l’AD. Pel fet que l’NO esdevé tòxic quan és produït en un entorn pro-oxidant, hem estudiat també l’impacte que el peroxinitrit té en l’ictus.
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Souza, Maria Sigride Thomé de. ""Leucoencefalopatia com substância branca evanescente: estudo clínico e de neuroimagem"." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/5/5138/tde-17042006-094726/.
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Leucoencefalopatia com substância branca evanescente é uma doença geneticamente determinada, causada por mutação no gene do eIF2B. A idade varia do período pré-natal até idade adulta, as manifestações geralmente são desencadeadas por trauma ou infecção. Os sintomas são variáveis, incluem ataxia cerebelar, espasticidade e relativa preservação cognitiva. Os achados de ressonância magnética (RM) são típicos e caracterizam-se por extenso comprometimento da substância branca. Estudamos 10 pacientes, com evolução súbita ou progressiva dos primeiros sintomas, entre 1 a 12 anos de idade. Ataxia e espasticidade estavam presentes em todos os pacientes e funções cognitivas relativamente preservadas. A clínica associada à RM, que demonstrava comprometimento difuso da substância branca, permitiu o diagnósticoLeukoencephalopathy with vanishing white matter is an inherited disorder caused by mutation in one of five subunits of eIF2B gene. Age of onset varies from prenatal to adulthood and manifestations are commonly triggered by trauma or infection. Symptoms are variable and include cerebellar ataxia and spasticity, with relative sparing of cognitive function. Magnetic resonance imaging (MRI) findings are typically characterized by widespread white matter abnormality. We studied 10 patients, with sudden or slowly progressive symptoms starting between 1-12 years of age. Ataxia and spasticity were present in all patients, and cognitive functions were relatively preserved. MRI studies demonstrated diffuse white matter abnormalities which, combined with clinical findings, allow diagnosis
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Sanséau, Doriane. "Étude du récepteur CD95 et de son rôle pro-inflammatoire dans le lupus." Thesis, Rennes 1, 2015. http://www.theses.fr/2015REN1S134.
Full textAbstract:
Le récepteur de mort CD95 participe à de nombreuses fonctions physiologiques en transmettant des signaux apoptotiques. Son ligand membranaire, CD95L, est principalement exprimé à la surface des lymphocytes et contrôle ainsi l’homéostasie cellulaire et l’élimination des cellules infectées ou transformées. Certaines situations pathologiques conduisent à une expression ectopique du CD95L par d’autres types cellulaires,associé à son clivage par des métalloprotéases (cl-CD95L). La forme soluble ainsi libérée perd sa capacité à transmettre l’apoptose mais déclenche l’activation de voies non-apoptotiques induisant l’inflammation dans des maladies inflammatoires chroniques comme le lupus érythémateux systémique (LES) ou encore les formes métastatiques du cancer du sein. Dans ces deux pathologies, de fortes quantités de cl-CD95L sont détectées dans le sérum de ces patients et ont été associés à la progression des pathologies. Dans le LES, nous établissons que cl-CD95L contribue au processus inflammatoireen favorisant la transmigration endothéliale des lymphocytes Th17. Cette migration cellulaire dépendante de CD95 nécessitele recrutement de la PLCγ1 sur le domaine juxta-membranaire de CD95 qui induitl’activation du signal calcique. Pour identifier d’autres partenaires moléculaires de CD95, une analyse protéomique a été réalisée et a permis d’identifier une association entre CD95 et la machinerie traductionnelle. Cette interaction nécessite le recrutement d’eIF4A1 au niveau du domaine juxta-membranaire de CD95. Nous avons par ailleurs montré que dans des lignées cancéreuses mammaires, eIF4A1 participe à la traduction de certaines protéines comme la sérine-thréonine kinase Akt pour faciliter l’activation de la voie de signalisation PI3K et la migration cellulaire induite par CD95. Cette étude a donc mis en évidence l’implication d’un nouveau domaine de CD95 dans l’induction des signaux non-apoptotiques. Ce domaine juxta-membranaire a été nommé CID pour « calcium inducing domain ». De plus, ce domaine fusionné à un peptide perméant provenant de la protéine TAT appelé TAT-CIDa montré son efficacité pour inhiber la migration lymphocytaire chez les souris lupiques et offre de nouvelles perspectives pour le développement de traitements améliorants les symptômes inflammatoires du LESThe death ligand CD95L, mainly expressed by immune cells, contributes to the elimination ofinfected and transformed cells. In pathological contexts, CD95L can be expressed by others cell types such as endothelial cells.CD95L can be cleaved by metalloproteases to generate a soluble CD95L (cl-CD95L) failing to trigger the apoptotic signaling pathwaybutinducing non-pro-apoptotic signaling pathways. cl-CD95L promotes inflammation in chronic inflammatory disorders such as systemic lupus erythematosus (SLE) and increases risks of metastatic dissemination in breast cancer patients. In SLE patients, we established that high amounts of cl-CD95L fuels inflammation by promoting endothelial transmigration of activated Th17 cells. This CD95-drivencell migration requires PLCγ1 recruitment by CD95 and the subsequentimplementation of the calcium signal. To identify in an exhaustive fashion, all molecular partners of CD95, a global proteomic analysis was undertaken. This TAP-Tag approach highlighted a strong association between the translational machinery and CD95. This analysis was confirmed by a two-hybrid approach revealingthat the translation initiation factor eIF4A1 directly interactedwith CD95.In breast cancer cells, we established that eIF4A1 was instrumental in the translation of certain genes such as Akt contributing to the implementation of the CD95-mediated PI3K signaling pathway and cell migration. In this study, we identifiedthe CD95 domaininvolved in the induction of the non-apoptotic signaling pathway. This domain was named CID for “calcium inducing domain”.Moreover, wegenerated a therapeutic molecule consisting of theCID fused to the cationic cell-penetrating HIV TAT domain. TAT-CID prevented the accumulation of Th17 cells in inflamed organs of lupus-prone mice and could turn out to be an original therapeutic molecule to alleviate clinical symptoms in SLE patients
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Rapone, Roberta. "Essential cytoplasmic role(s) of the histone lysine methyltransferase Setdb1 in post-transcriptional regulation of gene expression." Thesis, Université de Paris (2019-....), 2019. https://theses.md.univ-paris-diderot.fr/RAPONE_Roberta_va.pdf.
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Setdb1 est une «histone» lysine méthyltransférase (KMT) appartenant à la famille SUV39, l’une des principales machineries épigénétiques de répression des gènes. Setdb1 établit notamment la mono-, la di- et la tri- méthylation sur la lysine 9 de l'histone H3 (H3K9).Setdb1 est essentiel pour la survie, la pluripotence et l'auto-renouvellement des cellules souches embryonnaires de souris (mESCs); son knock-out est mortel au stade de la péri-implantation à 3,5 dpc chez la souris. Setdb1 est également nécessaire pour la différenciation de nombreux types de cellules progénitrices : spermatogenèse, neurogenèse, différenciation des chondrocytes et différenciation des muscles squelettiques. De plus, Setdb1 a été associé à plusieurs maladies: il est amplifié dans le mélanome et le cancer du poumon et il est dérégulé dans les cancers du foie, de la prostate, colorectal et du sein, dans la maladie de Huntington et la schizophrénie. Remarquablement, au-delà des histones, Setdb1 méthyle nombreux substrats non-histones, y compris UBF, p53, Akt, Tat et Ing2.Bien que Setdb1 ait toujours été associé à son rôle nucléaire, il s'avère que Setdb1 est la seule KMT de la famille SUV39 à avoir également une localisation cytoplasmique, dans plusieurs types de cellules, y compris les mESCs, les fibroblastes embryonnaires de souris (MEFs) et les cellules HeLa. Cependant, la fonction de Setdb1 dans le cytoplasme reste totalement inconnue. Pour étudier le rôle cytoplasmique de Setdb1, nous avons utilisé des cellules souches embryonnaires de souris (mESCs), dans lesquelles Setdb1 est essentiel. Nos résultats montrent que Setdb1 cytoplasmique est crucial pour la survie des mESCs: en effet, le nombre de cellules apoptotiques augmente après la perte de Setdb1 cytoplasmique. Nous avons constaté que le Setdb1 cytoplasmique affecte la synthèse de protéines dans les mESCs. Nous montrons en outre que le Setdb1 cytoplasmique interagit avec la protéine Trim71 spécifique de mESC (également appelée Lin41) et avec le facteur de traduction d'initiation eIF3c dans les mESC. Enfin, nous avons démontré que Setdb1 et Trim71 co-régulent ensemble la stabilité et la traduction des mARNs. Nos données actuelles mettent au jour la fonction cytoplasmique essentielle d'une lysine méthyltransférase appelée Setdb1, au début considérée comme étant spécifique uniquement des histones et apportent de nouvelles informations sur la régulation post-transcriptionnelle de l'expression génique médiée par un régulateur épigénétique fondamentalSetdb1 is a “histone” lysine methyltransferase (KMT) belonging to the SUV39 family that methylates lysine 9 of histone H3 (H3K9), one of the major epigenetic machineries mainly involved in gene repression. Notably, Setdb1 establishes mono-, di- and tri-methylation of H3K9. Setdb1, or Eset in mice, is essential for the survival, the pluripotency and the self-renewal of mouse embryonic stem cells (mESCs); Eset knockout is lethal at the peri-implantation stage at 3.5 dpc in mice. Setdb1 is also required for the differentiation of many progenitor cell types: spermatogenesis, neurogenesis, chondrocyte differentiation and skeletal muscle differentiation. Moreover, Setdb1 has been associated with several diseases: it is amplified in melanoma and lung cancer and it is dysregulated in liver, prostate, colorectal and breast cancers, Huntington disease and schizophrenia.Remarkably, beyond histones, Setdb1 methylates many non-histone substrates, such as UBF, p53, AKT, Tat and ING2 proteins. Although Setdb1 has been always associated with its nuclear role, it turns out that Setdb1 is the only H3K9 KMT to have also a cytoplasmic localization, in several cell types, including mESCs, mouse embryonic fibroblasts (MEFs) and HeLa cells. However, the function of Setdb1 in the cytoplasm remains totally unknown. To investigate Setdb1 cytoplasmic role, we have used mouse embryonic stem cells (mESCs), in which Setdb1 is essential. Our results show that cytoplasmic Setdb1 is crucial for the survival of mESCs: indeed, the number of apoptotic cells increases after the loss of cytoplasmic Setdb1. We found that cytoplasmic Setdb1 affects newly protein synthesis in mESCs. We further show that cytoplasmic Setdb1 interacts with mESCs-specific protein Trim71 (also called Lin41) and with the initiation translation factor eIF3c in mESCs. Finally, we reported that Setdb1 and Trim71 together co-regulate mRNA stability and translation. Our current data unravel the essential cytoplasmic function of Setdb1, for long time considered exclusively an “histone” lysine methyltransferase, and provide new insights into the post-transcriptional regulation of gene expression mediated by a fundamental epigenetic regulator
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Chu, Chia-Ying. "Molecular Mechanism of RNA-Mediated Gene Silencing in Human Cells: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/388.
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Small non-coding RNAs regulate gene expression at posttranscriptional level in eukaryotic cells. Two classes of such small (~21-25 nt) RNAs that have been extensively studied in gene silencing are short interfering RNAs (siRNAs) and microRNAs (miRNAs). RNA interference (RNAi) is process whereby double-stranded RNA induces the sequence-specific degradation of homologous mRNA. The RNAi machinery can also be programmed in human cells by introducing 21-nt siRNA duplexes that are assembled into RNA-induced silencing complexes (RISC). In this dissertation, systematic analysis of siRNAs with deletions at the passenger and/or guide strand reveals that a short RNAi trigger, 16-nt siRNA, induces potent RNAi in human cells. The 16-nt siRNA more effectively knocked down mRNA and protein levels than 19-nt siRNA when targeting the endogenous CDK9 gene. In vitro kinetic analysis of human RISC indicates that 16-nt siRNA has a higher RISC-loading capacity than 19-nt siRNA. These results suggest that 16-nt duplexes can be designed as potent triggers for RNAi.
RISC can be programmed by small interfering RNAs (siRISC) to cleave a perfectly complementary target mRNA, or endogenous microRNAs (miRISC) to inhibit translation by binding imperfectly matched sequences in the 3’-untranslated region (3’-UTR) of target mRNA. Both RISCs contain Argonaute2 (Ago2), which localizes to cytoplasmic mRNA processing P-bodies. This dissertation shows that RCK/p54, a DEAD box helicase, interacts with Ago2, in affinity-purified active siRISC or miRISC, facilitates formation of P-bodies. Depletion of RCK/p54 disrupted P-bodies and dispersed Ago2 throughout the cytoplasm, but did not significantly affect siRNA-mediated RNAi. Depleting RCK/p54 releases general and miRNA-induced translational repression. These findings imply that miRISC-mediated translation repression requires RCK/p54, also suggest that location of miRISC to P-bodies is not required for miRNA function, but is the consequence of translation repression.
To elucidate the function of RCK/p54 in miRNA-mediated gene silencing, analysis of a series of YFP-tagged RCK/p54 mutants reveals the motif required for P-body localization, interaction with Ago2, and/or facilitating the miRNA-mediated translation repression. Additionally, rabbit reticulocyte lysate system was used to recapitulate the miRISC function in a cell-free system and confirmed the requirement of RCK/p54 for miRNA function in vitro. Analysis of Ago2 distribution in the polysome profiling in RCK/p54-depleted cells, compared to that in normal cells, revealed that RCK/p54 facilitates miRISC by trapping it at translation initiation complex. These data suggest that interaction of RCK/p54 with Ago2 is involved in the repression of translation initiation of miRNA function.
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Bark, Abed El Rahim. "Conditional inactivation of EIF2B5 gene in oligodendrocytes or astrocytes cell lineages in adult mice." Thesis, Université de Paris (2019-....), 2019. https://theses.md.univ-paris-diderot.fr/BARK_Abed_El_Rahim_1_complete_20191218.pdf.
Full textAbstract:
Vanishing white matter disease (VWMD) ou childhood ataxia with central nervous system hypomyelination (CACH) est une maladie démyélinisante autosomale récessive qui présente un spectre phénotypique hétérogène et qui est causée par des mutations autosomales récessives touchant les gènes qui codent pour les cinq sous-unités du facteur d’initiation de la traduction EIF2B. Cette maladie provoque des dévastations et raréfactions de la substance blanche cérébrale avec une morphologie atypique des astrocytes et des oligodendrocytes d’aspect spumeux. Plusieurs modèles ont été développés pour comprendre la pathophysiologie de la maladie et pour essayer de trouver des stratégies thérapeutiques. Dans notre étude, nous avons établi deux nouveaux modèles murins. Dans le premier, nous avons ciblé le gène eif2b5 dans les oligodendrocytes en utilisant la forme inductible par Tamoxifen de le Cre (CreERT2) exprimée dans un locus de la protéine oligodendrocyte-spécifique protéolipoprotéine (PLP) ce qui permet la délétion du gène au moment précis dans les oligodendrocytes (Plp-Cre-ERT2/Eif2b5fl/fl). (i) Dans les souris femelles adultes, nous avons observé, neuf semaines après induction, une perte significative du poids accompagnée d’une paraplégie aigue ; (ii) Les tests de comportement ont montrés une déficience de la coordination motrice et de l’activité locomotrice avec une perte de la force musculaire ; (iii) Au niveau histologique, nous avons observé une baisse du nombre des oligodendrocytes matures olig2+/CC1+ avec persistance voir augmentation du pool des immatures. D’autre part, nous avons observé une augmentation des astrocytes GFAP+ et des microglies Iba1+ dans le cortex avec un aspect réactif des deux types cellulaires, en plus d’une perte neuronale. (iv) La microscopie électronique a montré une tendance qui reste non significative à la baisse du nombre des axones myélinisés ; (v) L’analyse en RNAseq des cellules triées (MACS) sur des cerveaux de souris adultes a révélé l’activation des voies du stress chronique exclusivement dans les oligodendrocytes immature (O4+) et la reprogrammation de l’expression des gènes favorisant le maintien des cellules immatures et l’activation des voies de la neuroinflammation lors de l’entrée dans la phase symptomatique. Dans le deuxième modèle, nous avons ciblé le gène eif2b5 dans les astrocytes en utilisant le même système avec la Cre exprimée dans un locus de la protéine Glutamate-Aspartate Transporter (Glast) ce qui permet la délétion du gène au moment précis dans les astrocytes (Glast-Cre-ERT2/Eif2b5fl/fl) ce qui a permis d’avoir deux phénotypes distincts résultant de la présence ou non d’une copie de Glast dans les souris (Glast-Cre-ERT2+/-/Eif2b5fl/fl avec une copie de Glast présente et Glast-Cre-ERT2-/-/Eif2b5fl/fl avec absence totale de Glast). (i) Chez les femelles Glast-Cre-ERT2+/-/Eif2b5fl/fl traitées au Tamoxifen, nous n’avons observé ni perte de poids ni paraplégie ; (ii) au niveau histologique, nous avons observé une augmentation des astrocytes GFAP+ et des microglies Iba1+ avec un aspect réactif des deux types cellulaires, alors que les oligodendrocytes et les neurones n’ont pas été affectés, ceci contrastant avec les souris Glast-Cre-ERT2-/-/Eif2b5fl/fl chez lesquelles nous avons observé (iii) une perte significative du poids accompagnée d’une paraplégie spastique à six semaines après induction; (iv) une augmentation des astrocytes GFAP+ et des microglies Iba1+ dans le cortex avec un aspect réactif des deux types cellulaires, en plus d’une baisse du nombre des oligodendrocytes matures olig2+/CC1+ et d’une perte neuronale. L’ensemble de ces travaux souligne la susceptibilité des cellules oligodendrocytes en particulier immature au stress du RE chronique induit par un déficit d’EIF2B. Les astrocytes nécessitent plusieurs facteurs associés pour entrainer une souffrance neurologique aigue. Ils sont peut être plus impliqués dans la phase développementale et chroniqueVanishing white matter disease (VWMD) or childhood ataxia with central nervous system hypomyelination (CACH) is an autosomal recessive demyelinating disorder with wide phenotypic heterogeneity caused by autosomal recessive mutations in the genes encoding the five subunits of the translation-initiation factor eIF2B. It causes cerebral white matter devastations and rarefactions with an atypical morphology of astrocytes and a “foamy” morphology of oligodendrocytes. Several animal models were developed to understand the pathophysiology of the disease and to investigate therapeutic strategies. In our studies, we established two new mouse models. In the first, we targeted the eif2b5 gene in oligodendrocytes in the tamoxifen inducible form of Cre (CreERT2) expressed in the locus of the oligodendrocyte-specific proteolipid protein (PLP) that allows precisely timed gene deletion in adult oligodendrocytes (Plp-Cre-ERT2/Eif2b5fl/fl). (i) In adult female mice, we observed after nine weeks of induction a significant loss of weight and acute paralysis; (ii) Behavioral tests, showed a deficiency in motor coordination and locomotor activity, and a loss of muscular strength; (iii) At the histological level, we observed a decrease of olig2/CC1 mature oligodendrocytes with persistence and even an increase in the pool of immature cells. On the other hand, an increase of number of GFAP+ astrocytes and Iba1+ microglia has been observed in cortex with an activated aspect of both cell types in addition to a neuronal loss; (iv) Electronic microscopy showed a non-significant but obvious tendency to decrease in the number of myelinated axons; (v) RNAseq analysis on cells sorted (MACS) from brains of adult mice showed the activation of pathways related to chronic stress, especially in the immature (O4+) oligodendrocytes and the reprogramming of the expression of genes favoring the maintaining of the immature cells and the activation of neuroinflammation pathways when going into the symptomatic phase. In the second model, we targeted the eif2b5 gene in astrocytes using the same system with the Cre expressed in the locus of the Glutamate-Aspartate Transporter (Glast) protein that allows precisely timed gene deletion in adult astrocytes (Glast-Cre-ERT2/Eif2b5fl/fl) which resulted in two distinct phenotypes related to the presence or not of a copy of the Glast locus in the resulting mice (Glast-Cre-ERT2+/-/Eif2b5fl/fl where one copy of Glast exists and Glast-Cre-ERT2-/-/Eif2b5fl/fl where no copy of Glast exists). (i) In the Glast-Cre-ERT2+/-/Eif2b5fl/fl TMX treated female mice, no weight loss nor paraplegia were observed; (ii) At the histological level, we observed an increase of number of GFAP+ astrocytes and Iba1+ microglia with an activated aspect of both cell types while no oligodendrocyte nor neuronal loss were seen in contrast to the Glast-Cre-ERT2-/-/Eif2b5fl/fl where (iii) significant loss of weight and paraplegia were observed at six weeks after induction; (iv) an increase of number of GFAP+ astrocytes and Iba1+ microglia has been observed with an activated aspect of both cell types in addition to mature oligodendrocyte and neuronal loss. This whole work underlines the susceptibility of oligodendrocyte cells, particularly the immature ones, to chronic ER stress induced by EIF2B deficit. The astrocytes require the association of many factors in order to cause acute neurologic distress. They might be more involved in the developmental and chronic phase