Eukaryotic translation initiation factor 5A (eIF5A) is the only known protein containing the unique amino acid, hypusine. eIF5A is present in all eukaryotic cells and is highly conserved, but its function is not well understood. The present investigation was undertaken to study the regulatory role of eIF5A in the induction of apoptosis in human cancer cell lines. Suppression of eIF5A1 using specific siRNA was shown to have no effect on the growth and proliferation of mammalian cells, although inhibition of the post-translational hypusination of eIF5A1 resulted in G1 phase cell cycle arrest. Treatment of HT-29 human colon adenocarcinoma cells with eIF5A1 siRNA did, however, reduce their sensitivity to pro-apoptotic stimuli including nitric oxide, Actinomycin D, proteaosome inhibition and serum starvation. Furthermore, over-expression of eIF5A1 in HT-29 cells or Hela S3 human cervical carcinoma cells using adenovirus constructs strongly induced apoptosis in a time-dependent manner. The pro-apoptotic effect of eIF5A1 appears to reflect at least in part its ability to activate the mitochondrial pathway of apoptosis in that its up-regulation resulted in dissipation of mitochondrial ∆Ψm, release of cytochrome c, activation of caspase 9 and caspase 3 and translocation of Bax from the cytosol to mitochondria. Similar effects were observed following treatment with eIF5A2, a second isoform of human eIF5A, and in addition eIF5A2 induced up-regulation of cleaved Bcl-2 which is thought to be pro-apoptotic. A mutant of eIF5A1 in which the conserved lysine, lysine50, that is post-translationally modified to hyusine was switched to alanine [eIF5A1(K50A)] also proved capable of inducing apoptosis by activating the mitochondrial pathway. As well, eIF5A1 and eIF5A1(K50A) both induced strong up-regulation of p73, a homolog of the tumor suppressor p53, in Hela S3 cells containing null p53. The finding that up-regulation of eIF5A1 also resulted in activation of caspase 8 indicates that it may be involved in regulation of the death receptor pathway of apoptosis as well. This contention is further supported by confocal microscopy studies indicating that, following its up-regulation, eIF5A1 localizes to the inner surface of the plasma membrane in a time-dependent manner that correlates temporally with the induction of apoptosis. eIF5A1 and its post-translationally modified forms were isolated by 2-dimensional Western blotting and sequenced by mass spectrometry. These analyses indicated that eIF5A1 containing unmodified lysine50 is the form of the protein that accumulates coincident with induction of apoptosis either by up-regulation of eIF5A1 or treatment with NO. These observations, together with the finding that eIF5A1(K50A) is capable of inducing apoptosis, indicate that it is the unhypusinated form of eIF5A1 that is apoptogenic.

Eukaryotic translation initiation factor 5A (eIF5A) is the only known protein containing the unique amino acid, hypusine. eIF5A is present in all eukaryotic cells and is highly conserved, but its function is not well understood. The present investigation was undertaken to study the regulatory role of eIF5A in the induction of apoptosis in human cancer cell lines. Suppression of eIF5A1 using specific siRNA was shown to have no effect on the growth and proliferation of mammalian cells, although inhibition of the post-translational hypusination of eIF5A1 resulted in G1 phase cell cycle arrest. Treatment of HT-29 human colon adenocarcinoma cells with eIF5A1 siRNA did, however, reduce their sensitivity to pro-apoptotic stimuli including nitric oxide, Actinomycin D, proteaosome inhibition and serum starvation. Furthermore, over-expression of eIF5A1 in HT-29 cells or Hela S3 human cervical carcinoma cells using adenovirus constructs strongly induced apoptosis in a time-dependent manner. The pro-apoptotic effect of eIF5A1 appears to reflect at least in part its ability to activate the mitochondrial pathway of apoptosis in that its up-regulation resulted in dissipation of mitochondrial ∆Ψm, release of cytochrome c, activation of caspase 9 and caspase 3 and translocation of Bax from the cytosol to mitochondria. Similar effects were observed following treatment with eIF5A2, a second isoform of human eIF5A, and in addition eIF5A2 induced up-regulation of cleaved Bcl-2 which is thought to be pro-apoptotic. A mutant of eIF5A1 in which the conserved lysine, lysine50, that is post-translationally modified to hyusine was switched to alanine [eIF5A1(K50A)] also proved capable of inducing apoptosis by activating the mitochondrial pathway. As well, eIF5A1 and eIF5A1(K50A) both induced strong up-regulation of p73, a homolog of the tumor suppressor p53, in Hela S3 cells containing null p53. The finding that up-regulation of eIF5A1 also resulted in activation of caspase 8 indicates that it may be involved in regulation of the death receptor pathway of apoptosis as well. This contention is further supported by confocal microscopy studies indicating that, following its up-regulation, eIF5A1 localizes to the inner surface of the plasma membrane in a time-dependent manner that correlates temporally with the induction of apoptosis. eIF5A1 and its post-translationally modified forms were isolated by 2-dimensional Western blotting and sequenced by mass spectrometry. These analyses indicated that eIF5A1 containing unmodified lysine50 is the form of the protein that accumulates coincident with induction of apoptosis either by up-regulation of eIF5A1 or treatment with NO. These observations, together with the finding that eIF5A1(K50A) is capable of inducing apoptosis, indicate that it is the unhypusinated form of eIF5A1 that is apoptogenic.

Made available in DSpace on 2014-06-11T19:30:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-11-26Bitstream added on 2014-06-13T19:19:50Z : No. of bitstreams: 1 gregio_apb_dr_arafcf.pdf: 2113336 bytes, checksum: b9ebdb1987717c610b020ac89d1288cf (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O provável fator de início de tradução 5A (eIF5A) é altamente conservado de arqueas a mamíferos e sofre uma modificação pós-traducional única e essencial, em que uma lisina específica é convertida em um resíduo de hipusina. Este fator já foi relacionado a início de tradução, transporte nucleocitoplasmático, decaimento de mRNA e proliferação celular, mas a função crítica de eIF5A na célula ainda não foi totalmente esclarecida. Ainda que o papel de eIF5A na tradução geral tenha sido questionado, dados recentes de nosso laboratório, incluindo os resultados deste trabalho, restabelecem uma função para eIF5A na tradução e evidenciam a sua atuação na etapa de elongação ao invés de início. Diante disso, o objetivo deste trabalho foi ampliar os estudos do papel de eIF5A na síntese proteica. Para isso, foram realizadas análises de interação genética e de perfil polissomal de linhagens que combinam o alelo mutante de eIF5A, tif51A-3, e de fatores envolvidos na etapa de início (eIF4E, eIF3 e eIF5B) ou elongação (eEF2) da tradução. As análises de perfil polissomal utilizando mutantes duplos de eIF5A e de fatores de início de tradução mostram uma diminuição do efeito de perda de polissomos característico de mutantes de início de tradução. Além disso, o perfil polissomal de mutantes de eIF5A é bastante semelhante ao do mutante de elongação. Foi também observado que a depleção de eIF5A provoca uma diminuição significativa na síntese proteica total e um aumento no tempo de trânsito ribossomal, confirmando os resultados obtidos nas análises de perfil polissomal. Também foi avaliada a influência de eIF5A no processo de elongação da tradução utilizando-se repórteres contendo a IRES do vírus CrPV, a qual é independente de todos os fatores de início de tradução conhecidos. Foi observada uma diminuição... / The putative eukaryotic translation initiation factor 5A (eIF5A) is highly conserved from archaea to mammals, essential for cell viability and it is the only cellular protein known to contain the essential amino acid hypusine. eIF5A has also been involved with nucleocitoplasmatic transport, mRNA decay and cell proliferation, however its critical function in the cell is not completely understood. Although a role for eIF5A in general translation has been questioned, our recent findings, including those described in this study, showed that eIF5A has a function in protein synthesis and it is related with the elongation step of translation instead of initiation. Therefore, the aim of this study was to investigate the role of eIF5A in protein synthesis. For this purpose, we tested genetic interactions and performed polysomal profile analysis of yeast strains that combine the eIF5A mutant tif51A-3 with translation initiation (eIF4E, eIF3 and eIF5B) or elongation (eEF2) mutants. The polysome profile analysis of the double mutants of eIF5A and canonical initiation mutants showed a decrease in polysome run-off. In addition, the polysome profile of eIF5A mutant is quite similar to that of the eEF2 mutant. Also, we observed that the depletion of eIF5A causes a significant decrease in total protein synthesis and an increase of the average ribosomal transit time. These results are in agreement with the polysome profile data. The influence of eIF5A in translation elongation was also evaluated using reporters containing the CrPV IRES, which is independent of all factors known to be involved in translation initiation. We observed a decrease in IRES activity in the eIF5A and eEF2 mutants, when compared to the wild type. In addition, two other conditional eIF5A mutants (tif51AK56A and tif51AQ22H/L93F) which produce stable eIF5A at the restrictive temperature were also analyzed... (Complete abstract click electronic access below)

Orientador: Cleslei Fernando Zanelli / Resumo: eIF5A é um fator de elongação da tradução conservado em arqueias e eucariotos, que interage com a subunidade maior do ribossomo 80S. É a única proteína que possui o aminoácido hipusina, formado pós-traducionalmente, essencial para sua função. eIF5A auxilia na tradução de proteínas que contêm motifs ricos em prolina, pois sequência repetida deste aminoácido pode causar parada do ribossomo em tradução devido à rigidez estrutural que forma. Na ausência de eIF5A funcional ocorre parada do ciclo celular em G1, na transição de G1 para S, tanto em Saccharomyces cerevisiae quanto em mamíferos, sugerindo papel essencial desta na tradução de proteínas importantes para a progressão do ciclo celular. Neste trabalho foi proposta a identificação das proteínas relevantes para a fase G1 que se encontram diferencialmente presentes durante desativação de eIF5A em S. cerevisiae. Foram utilizados dados de larga escala de perfil proteômico diferencial entre linhagens selvagens e mutantes de eIF5A, previamente obtido por nosso grupo, como ponto de partida para identificação das proteínas relevantes. A confirmação de níveis diferenciais destas proteínas foi realizada utilizando metodologia de Western blot após indução da desativação de eIF5A, utilizando o mutante sensível a temperatura hyp2-3. A desativação foi feita através do cultivo da linhagem mutante em temperatura semi-permissiva, que induz perda da função de eIF5A, porém mantendo viabilidade celular. Foi observado que a proteína Cka2 está di... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: eIF5A is a translation elongation factor conserved in archaea and eukaryotes, that interacts with the major subunit of the 80S ribosome. It is the only protein to contain the amino acid hypusine, formed post-translationally, essential to its function. eIF5A helps the translation of proteins that contain proline enriched motifs, as repeated sequence of this amino acid can cause ribosome stall during translation due to the rigid structure that it forms. In the absence of functional eIF5A occurs cell cycle arrest in G1, on the transition of G1 to S, both in Saccharomyces cerevisiae and mammals, suggesting an essential role of it on the translation of important proteins to the progression of the cell cycle. In this work, it was proposed the identification of relevant proteins to the G1 phase that are present differentially during deactivation of eIF5A in S. cerevisiae. It was used data of high-throughput differential proteomic profile between wild type strains and eIF5A mutant strains, previously obtained by our group, as a starting point to the identification of the relevant proteins. The confirmation of the differential levels of these proteins was made using Western blot methodology after induction of eIF5A deactivation, using the temperature-sensitive mutant hyp2-3. The deactivation was performed through cultivation of the mutant strain in semipermissive temperature, that induces loss of function of eIF5A but maintain cell viability. It was observed that the protein Cka2 is d... (Complete abstract click electronic access below) / Mestre

Although the ribosome is a very general catalyst, it cannot synthesize all protein sequences equally well. Certain proteins are capable of stalling the ribosome during their own synthesis. Stalling events are used by both prokaryotic and eukaryotic cells to regulate gene expression. Characterization of natural stalling peptides shows that only a few strategically placed amino acids are needed to inactivate the ribosome. These motifs share little sequence similarity suggesting that there are more stalling motifs yet to be discovered. Here we use two genetic selections in E. coli to discover novel stalling peptides and detail their subsequent characterization. Kinetic studies show that some of these nascent peptides dramatically inhibit rates of peptide release by release factors. We find that residues upstream of the minimal stalling motif can either enhance or suppress this effect. In other stalling motifs, such as polyproline sequences, peptidyl transfer to a subset of aminoacyl-tRNAs is inhibited. Translation factor EF-P alleviates pausing of the polyproline motifs, but has little or no effect on other stalling sequences. The EF-P ortholog eIF5A also alleviates pausing of polyproline sequences in yeast. Our studies show that short peptides sequences are capable of stalling the ribosome during elongation and termination through different mechanisms. These sequences are underrepresented in bacterial proteomes and show evidence of stalling on endogenous E. coli proteins.

Orientador: Cleslei Fernando Zanelli / Resumo: O fator de início de tradução 5A (eIF5A) é altamente conservado em arqueas e eucariotos e essencial para a viabilidade celular. eIF5A sofre uma modificação pós-traducional exclusiva e essencial para sua função, em que um resíduo específico de lisina é convertido em uma hipusina. Apesar eIF5A já ter sido relacionado com o início da tradução, uma quantidade crescente de estudos recentes têm estabelecido sua função na etapa de elongação da tradução, mais especificamente na elongação de sequências que são capazes de induzir um stalling (atraso ou parada) do ribossomo. Entretanto, existem ainda poucos trabalhos realizados com perfil proteômico na ausência de função de eIF5A, de maneira que atualmente pouco se sabe sobre as proteínas que têm sua tradução dependente de eIF5A. Desta forma, o presente projeto visa a busca de proteínas que têm sua tradução dependente de eIF5A através da comparação de perfil proteômico entre linhagens selvagens e mutantes de eIF5A em Saccharomyces cerevisiae. Para isto, utilizamos neste trabalho uma estratégia de perfil proteômico celular in vivo por fluorescência de GFP utilizando uma coleção de 4.156 linhagens, cada uma contendo uma ORF diferente em fusão com GFP no C-terminal, e uma proteína RFP (variante E2Crimson) constitutivamente produzida como normalizador, tanto no background selvagem (HYP2) como mutante para eIF5A (hyp2-3). Esta tese apresenta a análise dos dados de GFP/RFP e a validação desta análise utilizando-se western blot. Os resultados ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The translation factor 5A (eIF5A) is conserved and essential for cell viability. This is the only protein known to contain the amino acid residue hypusine, essential for eIF5A function, generated by a post-translational modification. Although it was initially suggested a function for eIF5A in the translation initiation, eIF5A has been demonstrated to have a role in translation elongation. More recent studies have established that eIF5A is necessary for the elongation of specific sequences, which are able to induce a ribosome stalling. Still, there are few studies with proteomic profile in the absence of eIF5A function and the proteins which syntheses are dependent on eIF5A are not well known. Thus, the present study aims to search for the proteins which syntheses are dependent on eIF5A by proteomic profile comparison between wild-type strains and eIF5A mutants in Saccharomyces cerevisiae. We present a proteomic profile for GFP fluorescence using a 4156 collection of strains, each one containing a different ORF fused to the C-terminal GFP and a protein RFP constitutively produced as normalizing, both in the wild and eIF5A mutant background. This thesis presents GFP / RFP data analysis and data validation using western blot. Our data using an in vivo proteome profile of the ORFs-GFP collection in a hyp2-3 mutant background demonstrating that yeast eIF5A shows several mitochondrial proteins downregulated in the eIF5A mutant. To confirm eIF5A involvement with mitochondrial functi... (Complete abstract click electronic access below) / Doutor

Eukaryotic cells tightly regulate metabolism in order to sustain normal processes. Dysregulation of cellular metabolism is associated with multiple diseases including cancer. Polyamine metabolism is a tightly regulated process that is co-opted by multiple cancers for selective growth advantages. Polyamines are small organic molecules with two or more amino groups attached, whose biosynthesis is initiated by ornithine decarboxylase (ODC). Although much is known regarding the effects of polyamine metabolism and ODC on cellular processes, little is known regarding the intracellular signaling events that are regulated by polyamines. Clinical studies demonstrated that the ODC inhibitor difluromethylornithine (DFMO) was an effective chemopreventative strategy causing a reduction of colon adenomas in patients with prior colon polyps. However, the molecular mechanisms leading to this reduction are unknown. This dissertation provides mechanistic insight into the biological roles of the polyamines and show that these amines are regulators of multiple non-coding RNAs involved in cellular responses including effects on the let-7 microRNA family. Moreover, the polyamine modified translation factor eIF5A is demonstrated to regulate the oncofetal factor LIN28. This work also indicates that polyamines regulate the mTOR pathway and suggests alternative signaling nodes for polyamine-mediated regulation of cellular processes. Overall, these findings support the notion that polyamines are oncometabolites that are targetable and serve as a promising approach to manipulate oncogenic signaling for cancer therapy.

Le stress oxydatif définit un phénomène cellulaire particulier caractérisé par un niveau élevé de molécules hautement réactives, essentiellement lié à l’utilisation de l’oxygène par les systèmes biologiques via la respiration. La dérégulation de l’état oxydatif de la cellule est à l’origine soit de processus d’adaptations efficaces (adaptation à l’altitude) soit de pathologies (AVC, infarctus). Ce travail de thèse s’est porté sur l’étude de deux nouvelles cibles pouvant induire une résistance/tolérance à la variation du stress oxydatif : la première est la protéine canal CFTR («Cystic Fibrosis Transmembrane conductance Regulator») et la seconde la voie d’activation d’eIF5A («eukaryotic Initiation translation Factor 5A»). Nous avons pu mettre en évidence que la protéine CFTR grâce à sa perméabilité au glutathion (l’antioxydant majoritaire cellulaire) est un modulateur de l’état oxydatif de la cellule, que ce soit lors de l’exposition à des agents cytotoxiques (cisplatine) ou lors de l’adaptation à des conditions hypoxiques chroniques. La deuxième cible identifiée est le facteur eIF5A qui est la seule protéine activée par la fixation d’un résidu hypusine. L’inhibition de cette modification post-traductionnelle protège les cellules d’une production d’espèces réactives induite par l’anoxie. Cette résistance à l’anoxie est accompagnée d’un profond remodelage métabolique et mitochondrial. Sur des modèles animaux d’ischémie (rein et cerveau), l’inhibition de l’activation d’eIF5A conduit à une protection des organes face à un manque d’oxygène. Ces études fondamentales ont des applications cliniques potentielles dans des pathologies humaines (infarctus, AVC, transplantation). / Oxidative stress represents a particular cellular condition, characterized by an intracellular increase in thereactive species level. These species are highly reactive towards biomolecules and result of oxygenconsumption by biological systems essentially through respiration. Deregulation of the cellular oxidativestate can initiate adaptive processes (as elevation adaptation) or several human pathologies (stroke,infarct). This thesis work has been devoted to the study of two news potential targets allowing atolerance/resistance towards disequilibrium of oxidative stress; the first one is CFTR, a channel protein(«Cystic Fibrosis Transmembrane conductance Regulator»), and the second one is the activation pathwayof the translation factor eIF5A («eukaryotic Initiation translation Factor 5A»). Based on the peculiaractivity of CFTR, consisting in the transport of glutathione, the major antioxidant of the cell, weevidenced the role of CFTR in the management of cellular oxidative state during cytotoxic drugexposure (cisplatin) or during adaptation to chronical hypoxia. The second target, eIF5A is the only oneprotein described as post-translationally modified by fixation of a hypusine residue. We demonstratedthat inhibition of eiF5A activation protect cells from reactive oxygen species generated during anoxia. Atcellular level, this protection is accompanied with deep metabolic and mitochondrial changes. Usinganimal models, we showed that inhibition of this eiF5A activation allows a tolerance against ischemicaccident in different organs (kidney and brain). These fundamentals results can have extensiveapplication in human clinical use (infarct, stroke, graft).

Plant development is complex and highly regulated. Tens of thousands of genes have been sequenced for the model plant <em>Arabidopsis thaliana</em>, yet few have been functionally annotated and characterized. This thesis describes the expression analysis and characterization of four genes in <em>Arabidopsis</em>. Three of these belong to the eukaryotic translation initiation factor 5A (eIF5A) gene family, and the fourth encodes diacylglycerol acyltransferase 1 (DGAT1). Putative roles for these genes in the development of <em>Arabidopsis thaliana</em> are described. <br /><br /> eIF5A is the only known protein to contain the amino acid hypusine. It has been demonstrated previously that eIF5A acts as a shuttle protein, moving specific mRNAs from the nucleus to the cytoplasm for translation. In <em>Arabidopsis thaliana</em> (At), there are three isoforms of eIF5A, and it is clear from the present study that they each have a unique temporal and spatial expression pattern. AteIF5A-1 and -2 are up-regulated during natural senescence and wounding/pathogenesis, respectively, and it is proposed that they regulate the onset of programmed cell death during these events. AteIF5A-3 is up-regulated in elongating meristem of the root, and it is proposed that this isoform is involved in cell growth. <br /><br /> Over-expression of the individual <em>AteIF5A</em> isoforms <em>in planta</em> resulted in pleiotropic phenotypes. When <em>AteIF5A-1</em> or <em>AteIF5A-2</em> was over-expressed, the phenotypes observed were indicative of their putative roles in the translation of proteins required for programmed cell death. When <em>AteIF5A-3</em> was over-expressed, the phenotypes were indicative of a role for this protein in the regulation of cell and tissue elongation. <br /><br /> Lipid analysis of rosette leaves from <em>Arabidopsis thaliana</em> revealed an accumulation of triacylglycerol with advancing leaf senescence coincident with an increase in the abundance and size of plastoglobuli. The terminal step in the biosynthesis of triacylglycerol in <em>Arabidopsis</em> is catalyzed by DGAT1. When gel blots of RNA isolated from rosette leaves at various stages of development were probed with the <em>Arabidopsis</em> EST clone, E6B2T7, which has been annotated as DGAT1, a steep increase in DGAT1 transcript levels was evident in the senescing leaves coincident with the accumulation of triacylglycerol. The increase in DGAT1 transcript correlated temporally with enhanced levels of DGAT1 protein detected immunologically. Two lines of evidence indicated that the triacylglycerol of senescing leaves is synthesized in chloroplasts and sequesters fatty acids released from the catabolism of thylakoid galactolipids. First, triacylglycerol isolated from senescing leaves proved to be enriched in hexadecatrienoic acid (16:3) and linolenic acid (18:3), which are normally present in thylakoid galactolipids. Second, DGAT1 protein in senescing leaves was found to be associated with chloroplast membranes. These findings collectively indicate that DGAT1 plays a role in senescence by sequestering fatty acids de-esterified from galactolipids into triacylglycerol.

As our body's guardian, the immune system maintains systemic health through removal of pathogens, damage and cancer. Ageing of the immune system is associated with compromised immune responses as well as decreased tumour surveillance and is therefore a key risk factor for major diseases in the elderly. Adaptive immune responses are mediated by T and B lymphocytes, and failure in adaptive immunity is a particular hallmark of the ageing organism. Here we show that autophagy is impaired in aged murine B lymphocytes, and loss of autophagy causes severely reduced B cell responses. Our data demonstrate that B cell senescence can be reversed in an autophagy-dependent manner by spermidine, a naturally occurring polyamine metabolite. Mechanistically, our study reveals that the translation factor eIF5A, that requires spermidine for its activation, regulates the expression of the master autophagy/lysosomal transcription factor TFEB. Importantly, we show in humans that spermidine, eIF5A and TFEB levels decrease with age and may serve as ageing biomarkers. Taken together our results indicate that the translational control of autophagy by eIF5A is dysregulated with ageing, and identify a novel pathway with therapeutic implications.