Functional significance of eIF5A and its hypusine modification in eukaryotes

The unusual basic amino acid, hypusine [N^ε-(4-amino-2-hydroxybutyl)-lysine], is a modified lysine with the addition of the 4-aminobutyl moiety from the polyamine spermidine. This naturally occurring amino acid is a product of a unique posttranslational modification that occurs in only one cellular protein, eukaryotic translation initiation factor 5A (eIF5A, eIF-5A). Hypusine is synthesized exclusively in this protein by two sequential enzymatic steps involving deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). The deoxyhypusine/hypusine synthetic pathway has evolved in archaea and eukaryotes, and eIF5A, DHS and DOHH are highly conserved suggesting a vital cellular function of eIF5A. Gene disruption and mutation studies in yeast and higher eukaryotes have provided valuable information on the essential nature of eIF5A and the deoxyhypusine/hypusine modification in cell growth and in protein synthesis. In view of the extraordinary specificity and functional significance of hypusine-containing eIF5A in mammalian cell proliferation, eIF5A and the hypusine biosynthetic enzymes are novel potential targets for intervention in aberrant cell proliferation.     

Posttranslational synthesis of hypusine: evolutionary progression and specificity of the hypusine modification

Summary. A naturally occurring unusual amino acid, hypusine [N ^ɛ-(4-amino-2-hydroxybutyl)-lysine] is a component of a single cellular protein, eukaryotic translation initiation factor 5A (eIF5A). It is a modified lysine with structural contribution from the polyamine spermidine. Hypusine is formed in a novel posttranslational modification that involves two enzymes, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). eIF5A and deoxyhypusine/hypusine modification are essential for growth of eukaryotic cells. The hypusine synthetic pathway has evolved in eukaryotes and eIF5A, DHS and DOHH are highly conserved, suggesting maintenance of a fundamental cellular function of eIF5A through evolution. The unique feature of the hypusine modification is the strict specificity of the enzymes toward its substrate protein, eIF5A. Moreover, DHS exhibits a narrow specificity toward spermidine. In view of the extraordinary specificity and the requirement for hypusine-containing eIF5A for mammalian cell proliferation, eIF5A and the hypusine biosynthetic enzymes present new potential targets for intervention in aberrant cell proliferation.     

eIF5A Promotes Translation Elongation,Polysome Disassembly and Stress Granule Assembly

Stress granules (SGs) are cytoplasmic foci at which untranslated mRNAs accumulate in cells exposed to environmental stress. We have identified ornithine decarboxylase (ODC), an enzyme required for polyamine synthesis, and eIF5A, a polyamine (hypusine)-modified translation factor, as proteins required for arsenite-induced SG assembly. Knockdown of deoxyhypusine synthase (DHS) or treatment with a deoxyhypusine synthase inhibitor (GC7) prevents hypusine modification of eIF5A as well as arsenite-induced polysome disassembly and stress granule assembly. Time-course analysis reveals that this is due to a slowing of stress-induced ribosome run-off in cells lacking hypusine-eIF5A. Whereas eIF5A only marginally affects protein synthesis under normal conditions, it is required for the rapid onset of stress-induced translational repression. Our results reveal that hypusine-eIF5A-facilitated translation elongation promotes arsenite-induced polysome disassembly and stress granule assembly in cells subjected to adverse environmental conditions.     

Effect of N 1-guanyl-1,7-diaminoheptane,an inhibitor of deoxyhypusine synthase,on endothelial cell growth,differentiation and apoptosis

An unusual amino acid, hypusine [N -(4-amino-2-hydroxybutyl)lysine], is formed post-translationally in a single cellular protein, the eukaryotic translation initiation factor 5A (eIF5A) by deoxyhypusine synthase and deoxyhypusine hydroxylase. Although eIF5A and its hypusine modification are essential for eukaryotic cell viability, the true physiological function of eIF5A is yet unknown. We have examined the effects of N ¹-guanyl-1,7-diaminoheptane (GC7), a potent inhibitor of deoxyhypusine synthase, on endothelial cell proliferation, differentiation and apoptosis. Upon treatment of human umbilical vein endothelial cells (HUVEC) with GC7, dose-dependent inhibition of hypusine formation and cellular proliferation was observed. GC7 at 10 M caused almost complete inhibition of cellular hypusine synthesis and led to cytostasis of HUVEC. Pretreatment of HUVEC with GC7 up to 50 M for 4 days had little effect on the attachment and differentiation of these cells on Matri-gel and did not cause induction of apoptosis. Instead, the GC7 pretreatment (96 h at 5–50 M) elicited protective effects against apoptotic death of HUVEC induced by serum starvation. These results suggest that eIF-5A may be involved in expression of proteins essential for apoptosis of endothelial cells as well as those for cellular proliferation.     

Biological Relevance and Therapeutic Potential of the Hypusine Modification System

Hypusine modification of the eukaryotic initiation factor 5A (eIF-5A) is emerging as a crucial regulator in cancer, infections, and inflammation. Although its contribution in translational regulation of proline repeat-rich proteins has been sufficiently demonstrated, its biological role in higher eukaryotes remains poorly understood. To establish the hypusine modification system as a novel platform for therapeutic strategies, we aimed to investigate its functional relevance in mammals by generating and using a range of new knock-out mouse models for the hypusine-modifying enzymes deoxyhypusine synthase and deoxyhypusine hydroxylase as well as for the cancer-related isoform eIF-5A2. We discovered that homozygous depletion of deoxyhypusine synthase and/or deoxyhypusine hydroxylase causes lethality in adult mice with different penetrance compared with haploinsufficiency. Network-based bioinformatic analysis of proline repeat-rich proteins, which are putative eIF-5A targets, revealed that these proteins are organized in highly connected protein-protein interaction networks. Hypusine-dependent translational control of essential proteins (hubs) and protein complexes inside these networks might explain the lethal phenotype observed after deletion of hypusine-modifying enzymes. Remarkably, our results also demonstrate that the cancer-associated isoform eIF-5A2 is dispensable for normal development and viability. Together, our results provide the first genetic evidence that the hypusine modification in eIF-5A is crucial for homeostasis in mammals. Moreover, these findings highlight functional diversity of the hypusine system compared with lower eukaryotes and indicate eIF-5A2 as a valuable and safe target for therapeutic intervention in cancer.     

The polyamine-derived amino acid hypusine: its post-translational formation in eIF-5A and its role in cell proliferation

Summary The unusual amino acid hypusine [N -(4-amino-2-hydroxybutyl)lysine] is a unique component of one cellular protein, eukaryotic translation initiation factor 5A (eIF-5A, old terminology, eIF-4D). It is formed posttranslationally and exclusively in this protein in two consecutive enzymatic reactions, (i) modification of a single lysine residue of the eIF-5A precursor protein by the transfer of the 4-aminobutyl moiety of the polyamine spermidine to its-amino group to form the intermediate, deoxyhypusine [N -(4-aminobutyl)lysine] and (ii) subsequent hydroxylation of this intermediate to form hypusine. The amino acid sequences surrounding the hypusine residue are strictly conserved in all eukaryotic species examined, suggesting the fundamental importance of this amino acid throughout evolution. Hypusine is required for the activity of eIF-5Ain vitro. There is strong evidence that hypusine and eIF-5A are vital for eukaryotic cell proliferation. Inactivation of both of the eIF-5A genes is lethal in yeast and the hypusine modification appears to be a requirement for yeast survival (Schnier et al., 1991 [Mol Cell Biol 11: 3105–3114]; Wöhl et al., 1993 [Mol Gen Genet 241: 305–311]). Furthermore, inhibitors of either of the hypusine biosynthetic enzymes, deoxyhypusine synthase or deoxyhypusine hydroxylase, exert strong anti-proliferative effects in mammalian cells, including many human cancer cell lines. These inhibitors hold potential as a new class of anticancer agents, targeting one specific eukaryotic cellular reaction, hypusine biosynthesis.     

The role of mammalian initiation factor eIF-4D and its hypusine modification in translation

Initiation factor eIF-4D functions late in the initiation pathway, apparently during formation of the first peptide bond. The factor is post-translationally modified at a specific lysine residue by reaction with spermidine and subsequent hydroxylation to form hypusine. A precursor form lacking hypusine is inactive in the assay for methionyl-puromycin synthesis, but activity is restored following in vitro modification to deoxyhypusine, thereby suggesting that the modification is essential for function. Since formylated methionyl-tRNA is less dependent on eIF-4D in the puromycin assay, we postulate that eIF-4D and its hypusine modification may stabilize charged Met-tRNA binding to the peptidyl transferase center of the 60S ribosomal subunit. Analysis of eIF-4D genes in yeast indicate that eIF-4D and its hypusine modification are essential for cell growth.     

Hypusine is required for a sequence-specific interaction of eukaryotic initiation factor 5A with postsystematic evolution of ligands by exponential enrichment RNA

Hypusine is formed through a spermidine-dependent posttranslational modification of eukaryotic initiation factor 5A (eIF-5A) at a specific lysine residue. The reaction is catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase. eIF-5A is the only protein in eukaryotes and archaebacteria known to contain hypusine. Although both eIF-5A and deoxyhypusine synthase are essential genes for cell survival and proliferation, the precise biological function of eIF-5A is unclear. We have previously proposed that eIF-5A may function as a bimodular protein, capable of interacting with protein and nucleic acid (Liu, Y. P., Nemeroff, M., Yan, Y. P., and Chen, K. Y. (1997) Biol. Signals 6, 166-174). Here we used the method of systematic evolution of ligands by exponential enrichment (SELEX) to identify the sequence specificity of the potential eIF-5A RNA targets. The post-SELEX RNA obtained after 16 rounds of selection exhibited a significant increase in binding affinity for eIF-5A with an apparent dissociation constant of 1 x 10(-7) m. The hypusine residue was found to be critical for this sequence-specific binding. The post-SELEX RNAs shared a high sequence homology characterized by two conserved motifs, UAACCA and AAUGUCACAC. The consensus sequence was determined as AAAUGUCACAC by sequence alignment and binding studies. BLAST analysis indicated that this sequence was present in > 400 human expressed sequence tag sequences. The C terminus of eIF-5A contains a cold shock domain-like structure, similar to that present in cold shock protein A (CspA). However, unlike CspA, the binding of eIF-5A to either the post-SELEX RNA or the 5'-untranslated region of CspA mRNA did not affect the sensitivity of these RNAs to ribonucleases. These data suggest that the physiological significance of eIF-5A-RNA interaction depends on hypusine and the core motif of the target RNA.     

Isolation and characterization of senescence-induced cDNAs encoding deoxyhypusine synthase and eucaryotic translation initiation factor 5A from tomato

Full-length cDNA clones encoding deoxyhypusine synthase (DHS) and eucaryotic initiation factor 5A (eIF-5A) have been isolated from a cDNA expression library prepared from tomato leaves (Lycopersicon esculentum, cv. Match) exposed to environmental stress. DHS mediates the first of two enzymatic reactions that activate eIF-5A by converting a conserved lysine to the unusual amino acid, deoxyhypusine. Recombinant protein obtained by expressing tomato DHS cDNA in Escherichia coli proved capable of carrying out the deoxyhypusine synthase reaction in vitro in the presence of eIF-5A. Of particular interest is the finding that DHS mRNA and eIF-5A mRNA show a parallel increase in abundance in senescing tomato flowers, senescing tomato fruit, and environmentally stressed tomato leaves exhibiting programmed cell death. Western blot analyses indicated that DHS protein also increases at the onset of senescence. It is apparent from previous studies with yeast and mammalian cells that hypusine-modified eIF-5A facilitates the translation of a subset of mRNAs mediating cell division. The present study provides evidence for senescence-induced DHS and eIF-5A in tomato tissues that may facilitate the translation of mRNA species required for programmed cell death.     

Hypusine formation in protein by a two-step process in cell lysates

The putative protein synthesis initiation factor eukaryotic initiation factor 4D (eIF-4D) is post-translationally modified by the polyamine spermidine, forming the rare amino acid hypusine from a lysine residue. The hypusine precursor, deoxyhypusine, was formed in crude cell lysates at pH 9.5 and converted to hypusine at pH 7.1. The modification occurred in eIF-4D, since the isoelectric points and molecular weights of the proteins modified in intact cells and lysates were indistinguishable. Only lysates from cells treated with alpha-difluoromethylornithine, to deplete endogenous polyamine pools, supported the formation of deoxyhypusine, suggesting that unmodified eIF-4D accumulated in spermidine deficient cells. Guazatine, an inhibitor of enzymes which form delta 1-pyrroline from spermidine, blocked deoxyhypusine formation in lysates by nearly 70% at 100 microM and completely at 1 mM. Other mammalian amine oxidase inhibitors had little or no effect on this reaction. Thus, deoxyhypusine formation in eIF-4D is catalyzed by a guazatine-sensitive enzyme with a basic pH optimum.     

Inhibition of EIF-5A prevents apoptosis in human cardiomyocytes after malaria infection

In this study, a determination of Troponin I and creatine kinase activity in whole-blood samples in a cohort of 100 small infants in the age of 2–5 years from Uganda with complicated Plasmodium falciparum malaria suggests the prevalence of cardiac symptoms in comparison to non-infected, healthy patients. Troponin I and creatine kinase activity increased during infection. Different reports showed that complicated malaria coincides with hypoxia in children. The obtained clinical data prompted us to further elucidate the underlying regulatory mechanisms of cardiac involvement in human cardiac ventricular myocytes. Complicated malaria is the most common clinical presentation and might induce cardiac impairment by hypoxia. Eukaryotic initiation factor 5A (eIF-5A) is involved in hypoxia induced factor (HIF-1α) expression. EIF-5A is a protein posttranslationally modified by hypusination involving catalysis of the two enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase. Treatment of human cardiomyocytes with GC7, an inhibitor of DHS, catalyzing the first step in hypusine biosynthesis led to a decrease in proinflammatory and proapoptotic myocardial caspase-1 activity in comparison to untreated cardiomyocytes. This effect was even more pronounced after co-administration of GC7 and GPI from P. falciparum simulating the pathology of severe malaria. Moreover, in comparison to untreated and GC7-treated cardiomyocytes, co-administration of GC7 and GPI significantly decreased the release of cytochrome C and lactate from damaged mitochondria. In sum, coadministration of GC7 prevented cardiac damage driven by hypoxia in vitro. Our approach demonstrates the potential of the pharmacological inhibitor GC7 to ameliorate apoptosis in cardiomyocytes in an in vitro model simulating severe malaria. This regulatory mechanism is based on blocking EIF-5A hypusination.     

The spermidine analogue GC7 (N1-guanyl-1,7-diamineoheptane) induces autophagy through a mechanism not involving the hypusination of eIF5A

The exogenous administration of spermidine promotes longevity in many model organisms. It has been proposed that this anti-age activity of spermidine is related to this polyamine’s ability to promote autophagy. Since spermidine is the substrate for the eIF5A post-translational modification by hypusination, we asked ourselves whether mature eIF5A may represent the link between spermidine and autophagy induction. To test this hypothesis, we inhibited the conversion of native eIF5A by a pharmacological approach, using the N1-guanyl-1,7-diamineoheptane (GC7), a spermidine analogue which competitively and reversibly inhibits deoxyhypusine synthase (DHS). In addition, we also employed genetic approaches by ablating both the eIF5A protein itself and DHS, the rate limiting enzyme catalyzing the conversion of lysine to hypusine. Collectively the data presented in this study demonstrate that the mature eIF5A (hypusinated form) is not involved in the autophagic pathway and that the inhibitor of DHS, GC7, produces off-target effect(s) resulting in marked induction of basal autophagy. These data are relevant in light of the fact that GC7 is considered a potent and selective inhibitor of DHS and is a potential candidate drug for cancer, diabetes and HIV therapy.     

Screening assay for the identification of deoxyhypusine synthase inhibitors

The 1st step in the posttranslational hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)lysine] modification of eukaryotic translation initiation factor 5A (eIF5A) is catalyzed by deoxyhypusine synthase (DHS). The eIF5A intermediate is subsequently hydroxylated by deoxyhypusine hydroxylase (DHH), thereby converting the eIF5A precursor into a biologically active protein. Depletion of eIF5A causes inhibition of cell growth, and the identification of eIF5A as a cofactor of the HIV Rev protein turns this host protein and therefore DHS into an interesting target for drugs against abnormal cell growth and/or HIV replication. The authors developed a 96-well format DHS assay applicable for the screening of DHS inhibitors. Using this assay, they demonstrate DHS inhibition by AXD455 (Semapimod, CNI-1493). This assay represents a powerful tool for the identification of new DHS inhibitors with potency against cancer and HIV.     

Effects of novel C-methylated spermidine analogs on cell growth via hypusination of eukaryotic translation initiation factor 5A

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.     

Protein-protein-interaction Network Organization of the Hypusine Modification System

Hypusine modification of eukaryotic initiation factor 5A (eIF-5A) represents a unique and highly specific post-translational modification with regulatory functions in cancer, diabetes, and infectious diseases. However, the specific cellular pathways that are influenced by the hypusine modification remain largely unknown. To globally characterize eIF-5A and hypusine-dependent pathways, we used an approach that combines large-scale bioreactor cell culture with tandem affinity purification and mass spectrometry: “bioreactor-TAP-MS/MS.” By applying this approach systematically to all four components of the hypusine modification system (eIF-5A1, eIF-5A2, DHS, and DOHH), we identified 248 interacting proteins as components of the cellular hypusine network, with diverse functions including regulation of translation, mRNA processing, DNA replication, and cell cycle regulation. Network analysis of this data set enabled us to provide a comprehensive overview of the protein-protein interaction landscape of the hypusine modification system. In addition, we validated the interaction of eIF-5A with some of the newly identified associated proteins in more detail. Our analysis has revealed numerous novel interactions, and thus provides a valuable resource for understanding how this crucial homeostatic signaling pathway affects different cellular functions.Cellular homeostasis is controlled by signaling networks that communicate through post-translational modifications (PTM)¹ of proteins, including phosphorylation, acetylation and methylation (1–3). These modifications are typically attached to various types of proteins by multiple independent enzymes, and thereby simultaneously regulate a wide range of protein functions. Consequently, most signaling pathways are highly redundant, enabling maintenance of cellular integrity even if the modification of a single signaling molecule is disrupted (4). A striking exception is hypusine. This essential PTM is limited to a single protein: the eukaryotic initiation factor 5A (eIF-5A) (5). Disruption of this PTM leads to growth arrest in proliferating eukaryotic cells and is fatal for the developing mammalian embryo (6, 7). During hypusine biosynthesis, the lysine residue at position 50 (Lys₅₀) in eIF-5A is converted into the unusual amino acid hypusine (N^ε-(4-amino-2-hydroxybutyl)lysine; depicted in Fig. 1A) (5). This process activates eIF-5A and is mediated by two enzymatic reactions: first, deoxyhypusine synthase (DHS) catalyzes the transfer of the 4-aminobutyl moiety of spermidine to the ε-amino group of Lys₅₀ to form an intermediate residue, deoxyhypusine (Dhp₅₀) (8). Subsequently, deoxyhypusine hydroxylase (DOHH) mediates the formation of hypusine (Hyp₅₀) by addition of a hydroxyl group to the deoxyhypusine residue (9). eIF-5A, DHS and DOHH are all essential for proliferation of higher eukaryotic cells (10, 11), and eIF-5A is strictly conserved throughout eukaryotic evolution (12).Open in a separate windowFig. 1.The hypusine modification and TAP fusion proteins employed in this study. A, The hypusine modification pathway and major proposed eIF-5A functions. B, Structure of the plasmid inserts coding for SG-tagged bait proteins. The amino acid positions of eIF-5A mutants are indicated in italic. SBP, streptavidin binding peptide. C, Metabolic incorporation of ³H-labeled spermidine into eIF-5A. Arrowheads indicate bands of SG-tagged and endogenous eIF-5A proteins. D, Anti-Myc-tag Western blot of cell lysates from retrovirally transduced Ba/F3 p210 cell lines for the quantification of constitutively expressed SG-tagged bait proteins. E, Representative TAP outputs for MS/MS analysis, after 1D PAGE separation and Coomassie staining. Separation distance varies from ∼2 to 4 cm.The eIF-5A protein has been proposed to promote various different cellular processes that potentially regulate proliferation, including translation initiation (13) and elongation (14) as well as nucleocytoplasmic transport of RNA or other cargoes (15, 16). Using inhibitors of DHS and DOHH or eIF-5A mutants deficient for hypusine modification, it has also been shown that this modification is a prerequisite of at least a subset of known eIF-5A functions (10, 11, 17, 18). The eIF-5A protein has also been implicated in numerous pathologic conditions including various types of cancer (19–23), β-cell inflammation (and therefore diabetes) (24) and HIV-1 infection (25). Human and rodent cells carry two highly homologous eIF-5A genes coding for distinct isoforms. Although eIF-5A1 is expressed at high levels throughout all tissues, eIF-5A2 is detectable only in a few embryonic tissues as well as adult testis, central nervous system (26), and cancer tissue (21, 22, 27–29).Although there have been ample reports suggesting eIF-5A is involved in translational control, the molecular mechanisms through which it ultimately influences cellular physiology and leads to disease remain unclear. Moreover, it remains equally possible that at least some of eIF-5A''s effects on cellular functions might not involve direct effects on translation. Also, there is no information available on whether the two isoforms of mammalian eIF-5A are functionally congruent.To address these fundamental questions systematically and comprehensively, we employed a bioreactor-based tandem affinity purification (TAP) approach followed by MS identification of purified protein complexes (“bioreactor-TAP-MS/MS”). To obtain a complete interaction map of the proteins involved in hypusine modification, we used this approach to identify interaction partners of both isoforms of eIF-5A, as well as the hypusine modification enzymes DHS and DOHH. In total, we identified 248 proteins that either directly interact with these bait proteins or are components of higher complexes containing the aforementioned proteins. Furthermore, we validated a subset of putative interaction partners of both eIF-5A isoforms, using Western blots of reciprocal TAP experiments, as well as a live-cell protein-fragment complementation assay (PCA). Our analysis provides a molecular framework for a detailed understanding on how this signal transduction pathway affects different crucial cellular functions.     

Hypusinated EIF5A as a feasible drug target for Advanced Medicinal Therapies in the treatment of pathogenic parasites and therapy-resistant tumors

Cancer drug resistance, in particular in advanced stages such as metastasis and invasion is an emerging problem. Moreover, drug resistance of parasites causing poverty-related diseases is an enormous, global challenge for drug development in the future. To circumvent this problem of increasing resistance, the development of either novel small compounds or Advanced Medicinal Therapies have to be fostered. Polyamines have many fundamental cellular functions like DNA stabilization, protein translation, ion channel regulation, autophagy, apoptosis and mostly important, cell proliferation. Consequently, many antiproliferative drugs can be commonly administered either in cancer therapy or for the treatment of pathogenic parasites. Most important for cell proliferation is the triamine spermidine, since it is an important substrate in the biosynthesis of the posttranslational modification hypusine in eukaryotic initiation factor 5A (EIF5A). To date, no small compound has been identified that directly inhibits the precursor protein EIF5A. Moreover, only a few small molecule inhibitors of the two biosynthetic enzymes, i.e. deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) have been functionally characterized. However, it is evident that only some of the compounds have been applied in translational approaches, i.e. in murine models to analyze the function of this modified protein in cell proliferation. In recent years, the pharmaceutical industry shifted from small molecules beyond traditional pharmacology to new tools and methods to treat disorders involving signaling deregulation. In this review, we evaluate translational approaches on inhibition of EIF5A hypusination in pathogenic parasites and therapy-resistant tumors and discuss its feasibility for an application in Advanced Medicinal Therapies.

     

Dramatic attenuation of hypusine formation on eukaryotic initiation factor 5A during senescence of IMR-90 human diploid fibroblasts

Deoxyhypusine synthase catalyzes the conversion of lysine to deoxyhypusine residue on the eukaryotic initiation factor 5A (eIF-5A) precursor using spermidine as the substrate. Subsequent hydroxylation of the deoxyhypusine residue completes hypusine formation on eIF-5A. Hypusine formation is one of the most specific polyamine-dependent biochemical events in eukaryotic cells. Although changes in polyamine metabolism have been demonstrated in human diploid fibroblasts during senescence (Chen and Chang, 1986, J. Cell. Physiol., 128:27–32.), it is unclear whether or not polyamine-dependent hypusine formation itself is an age-dependent biochemical event. In the present study, hypusine-forming activity was measured by a radiolabeling assay in cells whose polyamines have been depleted by prior treatment of α-difluoromethyl ornithine (DFMO). In addition, an in vitro cross-labeling assay was developed for simultaneous measurement of the deoxyhypusine synthase activity and protein substrate (eIF-5A precursor) amount. We showed that the hypusine-forming activity in low-passage presenescent IMR-90 cells [population doubling level (PDL) = 15–23, termed young cells] was prominently induced by serum whereas little or no hypusine-forming activity could be detected in late-passage senescent cells (PDL = 46–54, termed old cells). The striking difference in hypusine-forming activity between young and old cells was due to changes in both deoxyhypusine synthase activity and eIF-5A precursor amount in IMR-90 cells during senescence. However, Northern blot analysis showed no significant difference in the eIF-5A messenger RNA (mRNA) between young and old cells, suggesting that the age-dependent attenuation of eIF-5A precursor protein may be regulated at either translational or posttranslational level. J. Cell. Physiol. 170:248–254, 1997. © 1997 Wiley-Liss, Inc.