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Kuang Yu Chen - One of the best experts on this subject based on the ideXlab platform.
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thermal killing of human colon cancer cells is associated with the loss of eukaryotic Initiation Factor 5A
Journal of Cellular Physiology, 2009Co-Authors: Alexander Gosslau, David Lien Jao, Renee Butler, Alice Y C Liu, Kuang Yu ChenAbstract:Heat-induced cell death appears to be a cell-specific event. Chronic heat stress was lethal to human colon cancer cells (Caco-2, HT29, and HCT116), but not to normal diploid fibroblasts and other cancer cells (BJ-T, WI38, HeLa, ovarian 2008, WI38VA). Acute heat stress (45–51°C, 30 min) caused cell death of colon cancer cells during recovery at physiological temperature. Thermal killing of Caco-2 cells was not mediated via oxidative stress since Caco-2 cells were much more resistant than HeLa and other cancer cells to H2O2-induced cell death. Acute heat stress caused a striking loss of eukaryotic Initiation Factor 5A (eIF5A) in colon cancer cells, but not in HeLa and other normal or transformed human fibroblasts. The heat-induced loss of eIF5A is likely to be due to changes in the protein stability. The half-life of eIF5A was changed from >20 h to less than 30 min during the acute heat stress. Sequence analysis of the eIF5A gene from Caco-2 and HeLa cells did not reveal any difference, suggesting that the change in stability in Caco-2 cells was not due to any eIF5A mutation. Pretreatment of cells with protease inhibitors such as phenylmethyl sulfonyl fluoride (PMSF) partially blocked the heat-induced loss of eIF5A and prevented heat-induced cell death. In light of the essential role of eIF5A in cell survival and proliferation, our results suggest that the stability of eIF5A may have an important role in determining the fate of the particular cell type after severe heat stress. J. Cell. Physiol. 219: 485–493, 2009. © 2009 Wiley-Liss, Inc.
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tandem affinity purification revealed the hypusine dependent binding of eukaryotic Initiation Factor 5A to the translating 80s ribosomal complex
Journal of Cellular Biochemistry, 2006Co-Authors: David Lien Jao, Kuang Yu ChenAbstract:Eukaryotic Initiation Factor 5A (eIF5A) is the only protein in nature that contains hypusine, an unusual amino acid formed post-translationally in two steps by deoxyhypusine synthase and deoxyhypusine hydroxylase. Genes encodingeIF5Aordeoxyhypusinesynthaseareessentialforcellsurvivalandproliferation.Todeterminethephysiological function of eIF5A, we have employed the tandem affinity purification (TAP) method and mass spectrometry to search for and identify the potential eIF5A-interacting proteins. The TAP-tag was fused in-frame to chromosomal TIF51A gene and eIF5A-TAP fusion protein expressed at its natural level was used as the bait to fish out its interacting partners. At salt concentrations of 150 mM, deoxyhypusine synthase was the only protein bound to eIF5A. As salt concentrations were lowered to 125 mM or less, eIF5A interacted with a set of proteins, which were identified as the components of the 80S ribosome complex. The eIF5A-ribosome interaction was sensitive to RNase and EDTA treatments, indicating the requirementofRNAandthejoiningof40Sand60Sribosomalsubunitsfortheinteraction.Importantly,asinglemutationof hypusine to arginine completely abolished the eIF5A-ribosome interaction. Sucrose gradient sedimentation analysis of log versus stationary phase cells and eIF3 mutant strain showed that the endogenous eIF5A co-sedimented with the actively translating 80S ribosomes and polyribosomes in an RNase- and EDTA-sensitive manner. Our study demonstrates for the first time that eIF5A interacts in a hypusine-dependent manner with a molecular complex rather than a single protein, suggesting that the essential function of eIF5A is mostly likely mediated through its interaction with the actively translating ribosomes. J. Cell. Biochem. 97: 583-598, 2006. 2005 Wiley-Liss, Inc.
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rapid depletion of mutant eukaryotic Initiation Factor 5A at restrictive temperature reveals connections to actin cytoskeleton and cell cycle progression
Molecular Genetics and Genomics, 2006Co-Authors: Ishita Chatterjee, Stephane R Gross, Terri Goss Kinzy, Kuang Yu ChenAbstract:Eukaryotic Initiation Factor 5A (eIF5A) is the only protein in nature that contains hypusine, an unusual amino acid derived from the modification of lysine by spermidine. Two genes, TIF51A and TIF51B, encode eIF5A in the yeast Saccharomyces cerevisiae. In an effort to understand the structure-function relationship of eIF5A, we have generated yeast mutants by introducing plasmid-borne tif51A into a double null strain where both TIF51A and TIF51B have been disrupted. One of the mutants, tsL102A strain (tif51A L102A tif51aDelta tif51bDelta) exhibits a strong temperature-sensitive growth phenotype. At the restrictive temperature, tsL102A strain also exhibits a cell shape change, a lack of volume change in response to temperature increase and becomes more sensitive to ethanol, a hallmark of defects in the PKC/WSC cell wall integrity pathway. In addition, a striking change in actin dynamics and a complete cell cycle arrest at G1 phase occur in tsL102A cells at restrictive temperature. The temperature-sensitivity of tsL102A strain is due to a rapid loss of mutant eIF5A with the half-life reduced from 6 h at permissive temperature to 20 min at restrictive temperature. Phenylmethyl sulfonylfluoride (PMSF), an irreversible inhibitor of serine protease, inhibited the degradation of mutant eIF5A and suppressed the temperature-sensitive growth arrest. Sorbitol, an osmotic stabilizer that complement defects in PKC/WSC pathways, stabilizes the mutant eIF5A and suppresses all the observed temperature-sensitive phenotypes.
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Identification of mRNA that binds to eukaryotic Initiation Factor 5A by affinity co-purification and differential display.
Biochemical Journal, 2004Co-Authors: David Lien Jao, Kuang Yu ChenAbstract:Eukaryotic Initiation Factor 5A (eIF-5A) is the only protein in nature that contains hypusine, an unusual amino acid formed post-translationally by deoxyhypusine synthase and deoxyhypusine hydroxylase. Genetic and pharmacological evidence suggests that eIF-5A is essential for cell survival and proliferation. However, the precise function and interacting partners of eIF-5A remain unclear. We have shown previously that eIF-5A can bind to RRE (Rev-response element) and U6 RNA in vitro. Using SELEX (systematic evolution of ligands by exponential enrichment), we have also shown that eIF-5A is capable of binding to RNA in a sequence-specific manner [Xu and Chen (2001) J. Biol. Chem. 276, 2555-2561]. In the present paper, we show that the identification of mRNA species that bind to eIF-5A can be achieved by affinity co-purification and PCR differential display. Using this approach with three sets of anchoring and arbitrary primers, we have found 20 RNA sequences that co-purified specifically with eIF-5A. Five of them contained AAAUGU, the putative eIF-5A-interacting element that we identified previously using the SELEX method. Direct binding of the cloned RNA to eIF-5A could be demonstrated by electrophoretic mobility-shift assay. BLAST analysis revealed that the eIF-5A-interacting RNAs encode proteins such as ribosomal L35A, plasminogen activation inhibitor mRNA-binding protein, NADH dehydrogenase subunit and ADP-ribose pyrophosphatase. Some, however, encode hypothetical proteins. All the cloned RNAs have the potential to form extensive stem-loop structures.
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subcellular localization of the hypusine containing eukaryotic Initiation Factor 5A by immunofluorescent staining and green fluorescent protein tagging
Journal of Cellular Biochemistry, 2002Co-Authors: David Lien Jao, Kuang Yu ChenAbstract:Eukaryotic Initiation Factor 5A (eIF-5A) is the only protein in nature that contains hypusine, an unusual amino acid residue formed posttranslationally by deoxyhypusine synthase and deoxyhypusine hydroxylase. Although the eIF-5A gene is essential for cell survival and proliferation, the precise function and localization of eIF-5A remain unclear. In this study, we have determined the subcellular distribution of eIF-5A by indirect immunofluorescent staining and by direct visualization of green fluorescent protein tagged eIF-5A (GFP-eIF5A). Immunofluorescent staining of the formaldehyde-fixed cells showed that eIF-5A was present in both the nucleus and cytoplasm. Only the nuclear eIF-5A was resistant to Triton extraction. Direct visualization of GFP tagged eIF-5A in living cells revealed the same whole-cell distribution pattern. However, a fusion of an additional pyruvate kinase (PK) moiety into GFP-eIF-5A precluded the nuclear localization of GFP-PK-eIF-5A fusion protein. Fusion of the GFP-PK tag with three different domains of eIF-5A also failed to reveal any nuclear localization of the fusion proteins, suggesting the absence of receptor-mediated nuclear import. Using interspecies heterokaryon fusion assay, we could detect the nuclear export of GFP-Rev, but not of GFP-eIF-5A. The whole-cell distribution pattern of eIF-5A was recalcitrant to the treatments that included energy depletion, heat shock, and inhibition of transcription, translation, polyamine synthesis, or CRM1-dependent nuclear export. Collectively, our data indicate that eIF-5A gains nuclear entry via passive diffusion, but it does not undergo active nucleocytoplasmic shuttling. J. Cell. Biochem. 86: 590–600, 2002. © 2002 Wiley-Liss, Inc.
Myung Hee Park - One of the best experts on this subject based on the ideXlab platform.
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abstract 1998 alternative polyadenylation of eukaryotic Initiation Factor 5A 2 eif5A 2 mrnas examined by quadruplex single molecule rna fluorescencein situhybridization smrna fish
Cancer Research, 2016Co-Authors: Hans E Johansson, Raymund H Yin, Swati Mandal, Myung Hee ParkAbstract:Normal expression of the minor oncogene EIF5A2 in the hypusine pathway is restricted to brain and testis. However aberrant expression has been noted in multiple cancers, including ovarian gastric, colorectal and pancreatic, and artificial overexpression causes tumor growth in naked mice. Unlike cDNAs from the homologous gene EIF5A1, which are readily translated into protein upon transient transfection, EIF5A2-mRNAs only associate with monosomes without production of eIF5A-2 protein. Here we present data from multiple EIF5A2-positive cell lines on the steady state levels of the four different EIF5A2-mRNAs that arise through differential polyadenylation. While considering qPCR and RNA-Seq, we chose to apply Stellaris single molecule RNA fluorescence in situ hybridization with four unique probe sets, each reporting on the dependent A, B, C, or D segments. The probe sets were designed with multiple tiled singly labeled 18-22-mer oligonucleotides with balanced GC content, and hence similar hybridization characteristics, which in turn enabled the simultaneous quadruplex smRNA FISH. Stellaris RNA FISH leaves the cell intact, and also does not require RNA isolation or nucleic acid amplification that may introduce an otherwise hidden experimental bias. The single molecule spots revealed by widefield microscopy and subsequent image analysis co-localized with a high degree of confidence. Taking advantage of the added spatial dimension that RNA FISH affords, we also determined the localization of each mRNA variant and recorded the cell-to-cell variability and the actual range of mRNA copy numbers per cell. Our data is consistent with previous gene expression analysis data, but also provide actual counts of each mRNA variant. The results presented here have direct applications for the expression of the minor oncogene EIF5A2, and the regulated protein synthesis of its mRNAs, as well as of other mRNAs in polyamine and hypusine pathways. Further, the techniques and probes developed here can be directly transferred to the analysis of other mRNA variants that may arise through alternative transcription start site use, alternative splicing or alternative 3’ end processing in normal and aberrant cells. Citation Format: Hans E. Johansson, Raymund H. Yin, Swati Mandal, Myung-Hee Park. Alternative polyadenylation of eukaryotic Initiation Factor 5A-2 (eIF5A-2) mRNAs examined by quadruplex single molecule RNA fluorescence in situ hybridization (smRNA-FISH). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1998.
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a unique modification of the eukaryotic Initiation Factor 5A shows the presence of the complete hypusine pathway in leishmania donovani
PLOS ONE, 2012Co-Authors: Bhavna Chawla, Myung Hee Park, R R Kumar, Nidhi Tyagi, Gowri Subramanian, Narayanaswamy Srinivasan, Rentala MadhubalaAbstract:Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of an unusual amino acid hypusine (N-(sic)-(4-amino-2-hydroxybutyl) lysine), which is present on only one cellular protein, eukaryotic Initiation Factor 5A (eIF5A). We present here the molecular and structural basis of the function of DOHH from the protozoan parasite, Leishmania donovani, which causes visceral leishmaniasis. The L. donovani DOHH gene is 981 bp and encodes a putative polypeptide of 326 amino acids. DOHH is a HEAT-repeat protein with eight tandem repeats of alpha-helical pairs. Four conserved histidine-glutamate sequences have been identified that may act as metal coordination sites. A similar to 42 kDa recombinant protein with a His-tag was obtained by heterologous expression of DOHH in Escherichia coli. Purified recombinant DOHH effectively catalyzed the hydroxylation of the intermediate, eIF5A-deoxyhypusine (eIF5A-Dhp), in vitro. L. donovani DOHH (LdDOHH) showed similar to 40.6% sequence identity with its human homolog. The alignment of L. donovani DOHH with the human homolog shows that there are two significant insertions in the former, corresponding to the alignment positions 159-162 (four amino acid residues) and 174-183 (ten amino acid residues) which are present in the variable loop connecting the N- and C-terminal halves of the protein, the latter being present near the substrate binding site. Deletion of the ten-amino-acid-long insertion decreased LdDOHH activity to 14% of the wild type recombinant LdDOHH. Metal chelators like ciclopirox olamine (CPX) and mimosine significantly inhibited the growth of L. donovani and DOHH activity in vitro. These inhibitors were more effective against the parasite enzyme than the human enzyme. This report, for the first time, confirms the presence of a complete hypusine pathway in a kinetoplastid unlike eubacteria and archaea. The structural differences between the L. donovani DOHH and the human homolog may be exploited for structure based design of selective inhibitors against the parasite.
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Effects of novel C-methylated spermidine analogs on cell growth via hypusination of eukaryotic translation Initiation Factor 5A
Amino Acids, 2012Co-Authors: Mervi T Hyvonen, Tuomo A Keinanen, Alex R Khomutov, Jouko Vepsalainen, Leena Alhonen, Maxim Khomutov, Alina Simonian, Jong Hwan Park, Myung Hee ParkAbstract:The polyamines, putrescine, spermidine, and spermine, are ubiquitous multifunctional cations essential for cellular proliferation. One specific function of spermidine in cell growth is its role as a butylamine donor for hypusine synthesis in the eukaryotic Initiation Factor 5A (eIF5A). Here, we report the ability of novel mono -methylated spermidine analogs (α-MeSpd, β-MeSpd, γ-MeSpd, and ω-MeSpd) to function in the hypusination of eIF5A and in supporting the growth of DFMO-treated DU145 cells. We also tested them as substrates and inhibitors for deoxyhypusine synthase (DHS) in vitro. Of these compounds, α-MeSpd, β-MeSpd, and γ-MeSpd (but not ω-MeSpd) were substrates for DHS in vitro, while they all inhibited the enzyme reaction. As racemic mixtures, only α-MeSpd and β-MeSpd supported long-term growth (9–18 days) of spermidine-depleted DU145 cells, whereas γ-MeSpd and ω-MeSpd did not. The S -enantiomer of α-MeSpd, which supported long-term growth, was a good substrate for DHS in vitro, whereas the R -isomer was not. The long-term growth of DFMO-treated cells correlated with the hypusine modification of eIF5A by intracellular methylated spermidine analogs. These results underscore the critical requirement for hypusine modification in mammalian cell proliferation and provide new insights into the specificity of the deoxyhypusine synthase reaction.
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inactivation of eukaryotic Initiation Factor 5A eif5A by specific acetylation of its hypusine residue by spermidine spermine acetyltransferase 1 ssat1
Biochemical Journal, 2011Co-Authors: Jong Hwan Park, J E Folk, Jason A Deck, Anthony E Pegg, Masaaki Sokabe, Christopher S Fraser, Myung Hee ParkAbstract:eIF5A (eukaryotic translation Initiation Factor 5A) is the only cellular protein containing hypusine [ N ϵ -(4-amino-2-hydroxybutyl)lysine]. eIF5A is activated by the post-translational synthesis of hypusine and the hypusine modification is essential for cell proliferation. In the present study, we report selective acetylation of the hypusine and/or deoxyhypusine residue of eIF5A by a key polyamine catabolic enzyme SSAT1 (spermidine/spermine-N 1 -acetyltransferase 1). This enzyme normally catalyses the N 1 -acetylation of spermine and spermidine to form acetyl-derivatives, which in turn are degraded to lower polyamines. Although SSAT1 has been reported to exert other effects in cells by its interaction with other cellular proteins, eIF5A is the first target protein specifically acetylated by SSAT1. Hypusine or deoxyhypusine, as the free amino acid, does not act as a substrate for SSAT1, suggesting a macromolecular interaction between eIF5A and SSAT1. Indeed, the binding of eIF5A and SSAT1 was confirmed by pull-down assays. The effect of the acetylation of hypusine on eIF5A activity was assessed by comparison of acetylated with non-acetylated bovine testis eIF5A in the methionyl-puromycin synthesis assay. The loss of eIF5A activity by this SSAT1-mediated acetylation confirms the strict structural requirement for the hypusine side chain and suggests a possible regulation of eIF5A by hypusine acetylation/deacetylation.
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structural modeling and mutational analysis of yeast eukaryotic translation Initiation Factor 5A reveal new critical residues and reinforce its involvement in protein synthesis
FEBS Journal, 2008Co-Authors: Camila Arnaldo Olhe Dias, Myung Hee Park, Veridiana S P Cano, Suzana M Rangel, Luciano H Apponi, Mariana Carina Frigieri, J R C Muniz, Wanius Garcia, Richard Charles Garratt, Cleslei Fernando ZanelliAbstract:Eukaryotic translation Initiation Factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This Factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal α-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins – eIF5AK56A and eIF5AQ22H,L93F– and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.
Claudio M Costaneto - One of the best experts on this subject based on the ideXlab platform.
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evidences of a role for eukaryotic translation Initiation Factor 5A eif5A in mouse embryogenesis and cell differentiation
Journal of Cellular Physiology, 2010Co-Authors: Lucas T Parreirasesilva, Augusto Ducati Luchessi, Rui Curi, Eduardo B Oliveira, Rosana I Reis, Constance Oliver, Maria Celia Jamur, Ricardo Guelerman Pinheiro Ramos, Claudio M CostanetoAbstract:Eukaryotic translation Initiation Factor 5A (eIF5A) has a unique character: the presence of an unusual amino acid, hypusine, which is formed by post-translational modifications. Even before the identification of hypusination in eIF5A, the correlation between hypusine formation and protein synthesis, shifting cell proliferation rates, had already been observed. Embryogenesis is a complex process in which cellular proliferation and differentiation are intense. In spite of the fact that many studies have described possible functions for eIF5A, its precise role is under investigation, and to date nothing has been reported about its participation in embryonic development. In this study we show that eIF5A is expressed at all mouse embryonic post-implantation stages with increase in eIF5A mRNA and protein expression levels between embryonic days E10.5 and E13.5. Immunohistochemistry revealed the ubiquitous presence of eIF5A in embryonic tissues and organs at E13.5 day. Interestingly, stronger immunoreactivity to eIF5A was observed in the stomodeum, liver, ectoderm, heart, and eye, and the central nervous system; regions which are known to undergo active differentiation at this stage, suggesting a role of eIF5A in differentiation events. Expression analyses of MyoD, a myogenic transcription Factor, revealed a significantly higher expression from day E12.5 on, both at the mRNA and the protein levels suggesting a possible correlation to eIF5A. Accordingly, we next evidenced that inhibiting eIF5A hypusination in mouse myoblast C2C12 cells impairs their differentiation into myotubes and decreases MyoD transcript levels. Those results point to a new functional role for eIF5A, relating it to embryogenesis, development, and cell differentiation.
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involvement of eukaryotic translation Initiation Factor 5A eif5A in skeletal muscle stem cell differentiation
Journal of Cellular Physiology, 2009Co-Authors: Augusto Ducati Luchessi, Tavane D. Cambiaghi, Sandro M Hirabara, Rafael Herling Lambertucci, Leonardo R Silveira, Igor L Baptista, Anselmo Sigari Moriscot, Claudio M Costaneto, Rui CuriAbstract:The eukaryotic translation Initiation Factor 5A (eIF5A) contains a special amino acid residue named hypusine that is required for its activity, being produced by a post-translational modification using spermidine as substrate. Stem cells from rat skeletal muscles (satellite cells) were submitted to differentiation and an increase of eIF5A gene expression was observed. Higher content of eIF5A protein was found in satellite cells on differentiation in comparison to non-differentiated satellite cells and skeletal muscle. The treatment with N1-guanyl-1,7-diaminoheptane (GC7), a hypusination inhibitor, reversibly abolished the differentiation process. In association with the differentiation blockage, an increase of glucose consumption and lactate production and a decrease of glucose and palmitic acid oxidation were observed. A reduction in cell proliferation and protein synthesis was also observed. L-Arginine, a spermidine precursor and partial suppressor of muscle dystrophic phenotype, partially abolished the GC7 inhibitory effect on satellite cell differentiation. These results reveal a new physiological role for eIF5A and contribute to elucidate the molecular mechanisms involved in muscle regeneration.
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the n terminal region of eukaryotic translation Initiation Factor 5A signals to nuclear localization of the protein
Biochemical and Biophysical Research Communications, 2007Co-Authors: Lucas T Parreirasesilva, M Gomes, Eduardo B Oliveira, Claudio M CostanetoAbstract:The eukaryotic translation Initiation Factor 5A (eIF5A) is a ubiquitous protein of eukaryotic and archaeal organisms which undergoes hypusination, a unique post-translational modification. We have generated a polyclonal antibody against murine eIF5A, which in immunocytochemical assays in B16-F10 cells revealed that the endogenous protein is preferentially localized to the nuclear region. We therefore analyzed possible structural features present in eIF5A proteins that could be responsible for that characteristic. Multiple sequence alignment analysis of eIF5A proteins from different eukaryotic and archaeal organisms showed that the former sequences have an extended N-terminal segment. We have then performed in silico prediction analyses and constructed different truncated forms of murine eIF5A to verify any possible role that the N-terminal extension might have in determining the subcellular localization of the eIF5A in eukaryotic organisms. Our results indicate that the N-terminal extension of the eukaryotic eIF5A contributes in signaling this protein to nuclear localization, despite of bearing no structural similarity with classical nuclear localization signals.
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molecular modeling of the human eukaryotic translation Initiation Factor 5A eif5A based on spectroscopic and computational analyses
Biochemical and Biophysical Research Communications, 2006Co-Authors: Claudio M Costaneto, Lucas T Parreirasesilva, Eduardo B Oliveira, Roberto Ruller, Antonio Miranda, Laerte Oliveira, Richard J WardAbstract:The eukaryotic translation Initiation Factor 5A (eIF5A) is a protein ubiquitously present in archaea and eukarya, which undergoes a unique two-step post-translational modification called hypusination. Several studies have shown that hypusination is essential for a variety of functional roles for eIF5A, including cell proliferation and synthesis of proteins involved in cell cycle control. Up to now neither a totally selective inhibitor of hypusination nor an inhibitor capable of directly binding to eIF5A has been reported in the literature. The discovery of such an inhibitor might be achieved by computer-aided drug design based on the 3D structure of the human eIF5A. In this study, we present a molecular model for the human eIF5A protein based on the crystal structure of the eIF5A from Leishmania brasiliensis, and compare the modeled conformation of the loop bearing the hypusination site with circular dichroism data obtained with a synthetic peptide of this loop. Furthermore, analysis of amino acid variability between different human eIF5A isoforms revealed peculiar structural characteristics that are of functional relevance.
Dorian Bevec - One of the best experts on this subject based on the ideXlab platform.
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interaction of the hiv 1 rev coFactor eukaryotic Initiation Factor 5A with ribosomal protein l5
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Octavian Schatz, Dorian Bevec, Christiane Dascher, Michael Schebesta, Olaf Rosorius, Herbert Jaksche, Marika Dobrovnik, Joachim HauberAbstract:It has previously been shown that interaction of eukaryotic Initiation Factor 5A (eIF-5A) with the Rev trans-activator protein of HIV-1 mediates the transport of unspliced or incompletely spliced viral mRNAs across the nuclear envelope. Consequently, mutants of eIF-5A block Rev function and thereby replication of HIV-1 in trans, indicating that eIF-5A is a crucial protein that connects the viral Rev regulator with cellular RNA transport systems. Here we show that the ribosomal protein L5, which is the central protein component of the 5S rRNA export system, is a cellular interaction partner of eIF-5A. Functional studies demonstrate that overexpression of L5 protein significantly enhances Rev activity. Furthermore, Rev nuclear export activity is inhibited in human somatic cells by antibodies that recognize eIF-5A or L5. Our data suggest that the Rev export pathway shares components of a cellular transport system involved in the intracellular trafficking of polymerase III (5S rRNA) transcripts.
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eukaryotic Initiation Factor 5A activity and hiv 1 rev function
Neurosignals, 1997Co-Authors: Dorian Bevec, Joachim HauberAbstract:Eukaryotic Initiation Factor 5A (eIF-5A) is the only cellular protein known to contain the unusual amino acid hypusine, a modification that appears to be required for cell proliferation. This hypusine
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induced gene expression of the hypusine containing protein eukaryotic Initiation Factor 5A in activated human t lymphocytes
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Dorian Bevec, Friedrich Lottspeich, Hannelore Klier, Wolfgang Holter, Erwin Tschachler, Peter Valent, Thomas Baumruker, Joachim HauberAbstract:Abstract The hypusine-containing protein eukaryotic Initiation Factor 5A (eIF-5A) is a cellular coFactor critically required for the function of the Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1). eIF-5A localizes in the nuclear and cytoplasmic compartments of mammalian cells, suggesting possible activities on the level of regulated mRNA transport and/or protein translation. In this report we show that eIF-5A gene expression is constitutively low but inducible with T-lymphocyte-specific stimuli in human peripheral blood mononuclear cells (PBMCs) of healthy individuals. In contrast, eIF-5A is constitutively expressed at high levels in human cell lines as well as in various human organs. Comparison of eIF-5A levels in the PBMCs of uninfected and HIV-1-infected donors shows a significant upregulation of eIF-5A gene expression in the PBMCs of HIV-1 patients, compatible with a possible role of eIF-5A in HIV-1 replication during T-cell activation.
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the genomic structure encoding human Initiation Factor eif 5A
Gene, 1994Co-Authors: Karl Koettnitz, Thomas Baumruker, Joachim Hauber, Barbara Kappel, Dorian BevecAbstract:Abstract A genomic clone encoding the human eukaryotic Initiation Factor 5A (eIF-5A) was isolated and its entire nucleotide sequence was determined. The whole eIF-5A coding region is not interrupted by introns. The functional eIF-5A gene is highly homologous to the corresponding complementary DNA. One single 1.4-kb transcript thereof is expressed in human cell lines. Furthermore, we also isolated and sequenced two additonal eIF-5A -related sequences which are, by expression and sequence analyses, identified as pseudogenes of the functional eIF-5A . The sequence homology between these pseudogenes and the functional eIF-5A is 71 and 87%.
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eukaryotic Initiation Factor 5A is a cellular target of the human immunodeficiency virus type 1 rev activation domain mediating trans activation
Journal of Cell Biology, 1993Co-Authors: M Ruhl, Herbert Jaksche, M Himmelspach, G M Bahr, F Hammerschmid, B Wolff, H Aschauer, G K Farrington, H Probst, Dorian BevecAbstract:Expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the presence of the viral trans-activator protein Rev. Rev is localized in the nucleus and binds specifically to the Rev response element (RRE) sequence in viral RNA. Furthermore, the interaction of the Rev activation domain with a cellular coFactor is essential for Rev function in vivo. Using cross-linking experiments and Biospecific Interaction Analysis (BIA) we identify eukaryotic Initiation Factor 5A (eIF-5A) as a cellular Factor binding specifically to the HIV-1 Rev activation domain. Indirect immunofluorescence studies demonstrate that a significant fraction of eIF-5A localizes to the nucleus. We also provide evidence that Rev transactivation is functionally mediated by eIF-5A in Xenopus oocytes. Furthermore, we are able to block Rev function in mammalian cells by antisense inhibition of eIF-5A gene expression. Thus, regulation of HIV-1 gene expression by Rev involves the targeting of RRE-containing RNA to components of the cellular translation Initiation complex.
Augusto Ducati Luchessi - One of the best experts on this subject based on the ideXlab platform.
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Characterization of eukaryotic translation Initiation Factor 5A isoform A (752.13)
The FASEB Journal, 2014Co-Authors: Leticia Tamborlin, Karina Pereira, Isadora Almeida, Letícia Meneguello, Tavane D. Cambiaghi, Augusto Ducati LuchessiAbstract:The eukaryotic translation Initiation Factor 5A (EIF5A) protein is highly conserved in eukaryotes. Some studies shown that eIF5A has important roles in cell proliferation control and HIV-1 replicat...
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evidences of a role for eukaryotic translation Initiation Factor 5A eif5A in mouse embryogenesis and cell differentiation
Journal of Cellular Physiology, 2010Co-Authors: Lucas T Parreirasesilva, Augusto Ducati Luchessi, Rui Curi, Eduardo B Oliveira, Rosana I Reis, Constance Oliver, Maria Celia Jamur, Ricardo Guelerman Pinheiro Ramos, Claudio M CostanetoAbstract:Eukaryotic translation Initiation Factor 5A (eIF5A) has a unique character: the presence of an unusual amino acid, hypusine, which is formed by post-translational modifications. Even before the identification of hypusination in eIF5A, the correlation between hypusine formation and protein synthesis, shifting cell proliferation rates, had already been observed. Embryogenesis is a complex process in which cellular proliferation and differentiation are intense. In spite of the fact that many studies have described possible functions for eIF5A, its precise role is under investigation, and to date nothing has been reported about its participation in embryonic development. In this study we show that eIF5A is expressed at all mouse embryonic post-implantation stages with increase in eIF5A mRNA and protein expression levels between embryonic days E10.5 and E13.5. Immunohistochemistry revealed the ubiquitous presence of eIF5A in embryonic tissues and organs at E13.5 day. Interestingly, stronger immunoreactivity to eIF5A was observed in the stomodeum, liver, ectoderm, heart, and eye, and the central nervous system; regions which are known to undergo active differentiation at this stage, suggesting a role of eIF5A in differentiation events. Expression analyses of MyoD, a myogenic transcription Factor, revealed a significantly higher expression from day E12.5 on, both at the mRNA and the protein levels suggesting a possible correlation to eIF5A. Accordingly, we next evidenced that inhibiting eIF5A hypusination in mouse myoblast C2C12 cells impairs their differentiation into myotubes and decreases MyoD transcript levels. Those results point to a new functional role for eIF5A, relating it to embryogenesis, development, and cell differentiation.
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involvement of eukaryotic translation Initiation Factor 5A eif5A in skeletal muscle stem cell differentiation
Journal of Cellular Physiology, 2009Co-Authors: Augusto Ducati Luchessi, Tavane D. Cambiaghi, Sandro M Hirabara, Rafael Herling Lambertucci, Leonardo R Silveira, Igor L Baptista, Anselmo Sigari Moriscot, Claudio M Costaneto, Rui CuriAbstract:The eukaryotic translation Initiation Factor 5A (eIF5A) contains a special amino acid residue named hypusine that is required for its activity, being produced by a post-translational modification using spermidine as substrate. Stem cells from rat skeletal muscles (satellite cells) were submitted to differentiation and an increase of eIF5A gene expression was observed. Higher content of eIF5A protein was found in satellite cells on differentiation in comparison to non-differentiated satellite cells and skeletal muscle. The treatment with N1-guanyl-1,7-diaminoheptane (GC7), a hypusination inhibitor, reversibly abolished the differentiation process. In association with the differentiation blockage, an increase of glucose consumption and lactate production and a decrease of glucose and palmitic acid oxidation were observed. A reduction in cell proliferation and protein synthesis was also observed. L-Arginine, a spermidine precursor and partial suppressor of muscle dystrophic phenotype, partially abolished the GC7 inhibitory effect on satellite cell differentiation. These results reveal a new physiological role for eIF5A and contribute to elucidate the molecular mechanisms involved in muscle regeneration.
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Insights on eukaryotic translation Initiation Factor 5A (eIF5A) in the brain and aging.
Brain research, 2008Co-Authors: Augusto Ducati Luchessi, Tavane D. Cambiaghi, Claudio M. Costa-neto, Adilson S. Alves, Lucas T. Parreiras-e-silva, Luiz R.g. Britto, Rui CuriAbstract:Long-term memory, a persistent form of synaptic plasticity, requires translation of a subset of mRNA present in neuronal dendrites during a short and critical period through a mechanism not yet fully elucidated. Western blotting analysis revealed a high content of eukaryotic translation Initiation Factor 5A (eIF5A) in the brain of neonatal rats, a period of intense neurogenesis rate, differentiation and synaptic establishment, when compared to adult rats. Immunohistochemistry analysis revealed that eIF5A is present in the whole brain of adult rats showing a variable content among the cells from different areas (e.g. cortex, hippocampus and cerebellum). A high content of eIF5A in the soma and dendrites of Purkinje cells, key neurons in the control of motor long-term memory in the cerebellum, was observed. Detection of high eIF5A content was revealed in dendritic varicosities of Purkinje cells. Evidence is presented herein that a reduction of eIF5A content is associated to brain aging.
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Inhibition of eukaryotic translation Initiation Factor 5A (eIF5A) hypusination impairs melanoma growth
Cell biochemistry and function, 2006Co-Authors: Miriam Galvonas Jasiulionis, Augusto Ducati Luchessi, Andreia G. Moreira, Pedro P. C. Souza, Ana Paula M. Suenaga, Mariangela Correa, Carlos Alberto De Souza Costa, Rui Curi, Claudio M. Costa-netoAbstract:The eukaryotic translation Initiation Factor 5A (eIF5A) undergoes a specific post-translational modification called hypusination. This modification is required for the functionality of this protein. The compound N1-guanyl-1,7-diaminoheptane (GC7) is a potent and selective inhibitor of deoxyhypusine synthase, which catalyses the first step of eIF5A hypusination process. In the present study, the effects of GC7 on cell death were investigated using two cell lines: melan-a murine melanocytes and Tm5 murine melanoma. In vitro treatment with GC7 increased by 3-fold the number of cells presenting DNA fragmentation in Tm5 cells. Exposure to GC7 also decreased viability to both cell lines. This study also describes, for the first time, the in vivo antitumour effect of GC7, as indicated by impaired melanoma growth in C57BL/6 mice.