Reversal of the deoxyhypusine synthesis reaction. Generation of spermidine or homospermidine from deoxyhypusine by deoxyhypusine synthase

Deoxyhypusine synthase catalyzes the first step in hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine) synthesis in a single cellular protein, eIF5A precursor. The synthesis of deoxyhypusine catalyzed by this enzyme involves transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in the eIF5A precursor protein to form a deoxyhypusine-containing eIF5A intermediate, eIF5A(Dhp). We recently discovered the efficient reversal of deoxyhypusine synthesis. When eIF5A([3H]Dhp), radiolabeled in the 4-aminobutyl portion of its deoxyhypusine residue, was incubated with human deoxyhypusine synthase, NAD, and 1,3-diaminopropane, [3H]spermidine was formed by a rapid transfer of the radiolabeled 4-aminobutyl side chain of the [3H]deoxyhypusine residue to 1,3-diaminopropane. No reversal was observed with [3H]hypusine protein, suggesting that hydroxylation at the 4-aminobutyl side chain of the deoxyhypusine residue prevents deoxyhypusine synthase-mediated reversal of the modification. Purified human deoxyhypusine synthase also exhibited homospermidine synthesis activity when incubated with spermidine, NAD, and putrescine. Thus it was found that [14C]putrescine can replace eIF5A precursor protein as an acceptor of the 4-aminobutyl moiety of spermidine to form radiolabeled homospermidine. The Km value for putrescine (1.12 mM) as a 4-aminobutyl acceptor, however, is much higher than that for eIF5A precursor (1.5 microM). Using [14C]putrescine as an acceptor, various spermidine analogs were evaluated as donor substrates for human deoxyhypusine synthase. Comparison of spermidine analogs as inhibitors of deoxyhypusine synthesis, as donor substrates for synthesis of deoxyhypusine (or its analog), and for synthesis of homospermidine (or its analog) provides new insights into the intricate specificity of this enzyme and versatility of the deoxyhypusine synthase reaction.     

Molecular cloning and functional expression of bovine deoxyhypusine hydroxylase cDNA and homologs

Deoxyhypusine hydroxylase is the second of the two enzymes that catalyzes the maturation of eukaryotic initiation factor 5A (eIF5A). The mature eIF5A is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine (N(epsilon)-(4-amino-2(R)-hydroxybutyl)lysine). Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Here, we describe the cloning and characterization of bovine deoxyhypusine hydroxylase cDNA and its homologs. The deduced bovine deoxyhypusine hydroxylase protein is 87% identical to human enzyme and 45% identical to yeast enzyme. The overexpressed enzyme showed activity in catalyzing the hydroxylation of the deoxyhypusine residue in the eIF5A intermediate. An amino acid substitution from Glu 57 to Gly located at one of the four conserved His-Glu (HE) pairs, the potential metal coordination sites, resulted in severe reduction of deoxyhypusine hydroxylase activity. A deletion at the HEAT-repeats 1-3 resulted in complete losses of deoxyhypusine hydroxylase activity.     

Comparison of the activities of variant forms of eIF-4D. The requirement for hypusine or deoxyhypusine.

Eukaryotic protein synthesis initiation factor 4D (eIF-4D) (current nomenclature, eIF-5A) contains the unique amino acid hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). The first step in hypusine biosynthesis, i.e. the formation of the intermediate, deoxyhypusine (N epsilon-(4-aminobutyl)lysine), was carried out in vitro using spermidine, deoxyhypusine synthase, and ec-eIF-4D(Lys), an eIF-4D precursor prepared by over-expression of human eIF-4D cDNA in Escherichia coli. In a parallel reaction, using N-(3-aminopropyl)cadaverine in place of spermidine, a variant form of eIF-4D containing homodeoxyhypusine (N epsilon-(5-aminopentyl)lysine) was prepared. Evidence that N-(3-aminopropyl)cadaverine can also act as the amine substrate for deoxyhypusine synthase in intact cells was obtained by incubating putrescine- and spermidine-depleted Chinese hamster ovary cells with [3H]cadaverine. In these cells, in which [3H]cadaverine is readily converted to N-(3-aminopropyl) [3H]cadaverine, small amounts of [3H]homodeoxyhypusine and another 3H-labeled compound, presumed to be N epsilon-(5-amino-2-hydroxy[3H]pentyl)lysine, were found. eIF-4D stimulates methionyl-puromycin synthesis, an in vitro model assay for translation initiation. Whereas the unmodified precursor ec-eIF-4D(Lys) appeared inactive, the deoxyhypusine-containing form provided a significant degree of stimulation. The variant form containing homodeoxyhypusine, on the other hand, showed little or no activity. These findings emphasize the importance of hypusine or deoxyhypusine for the biological activity of eIF-4D and demonstrate the influence of both the length and chemical nature of its amino alkyl side chain.     

Inactivation of eukaryotic initiation factor 5A (eIF5A) by specific acetylation of its hypusine residue by spermidine/spermine acetyltransferase 1 (SSAT1)

eIF5A (eukaryotic translation initiation factor 5A) is the only cellular protein containing hypusine [N?-(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-N1-acetyltransferase 1). This enzyme normally catalyses the N1-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.     

The post-translational synthesis of a polyamine-derived amino acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF5A)

The eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the unique polyamine-derived amino acid, hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine]. Hypusine is formed in eIF5A by a novel post-translational modification reaction that involves two enzymatic steps. In the first step, deoxyhypusine synthase catalyzes the cleavage of the polyamine spermidine and transfer of its 4-aminobutyl moiety to the epsilon-amino group of one specific lysine residue of the eIF5A precursor to form a deoxyhypusine intermediate. In the second step, deoxyhypusine hydroxylase converts the deoxyhypusine-containing intermediate to the hypusine-containing mature eIF5A. The structure and mechanism of deoxyhypusine synthase have been extensively characterized. Deoxyhypusine hydroxylase is a HEAT-repeat protein with a symmetrical superhelical structure consisting of 8 helical hairpins (HEAT motifs). It is a novel metalloenzyme containing tightly bound iron at the active sites. Four strictly conserved His-Glu pairs were identified as iron coordination sites. The structural fold of deoxyhypusine hydroxylase is entirely different from those of the other known protein hydroxylases such as prolyl 4-hydroxylase and lysyl hydroxylases. The eIF5A protein and deoxyhypusine/hypusine modification are essential for eukaryotic cell proliferation. Thus, hypusine synthesis represents the most specific protein modification known to date, and presents a novel target for intervention in mammalian cell proliferation.     

Evaluation of the metal ion requirement of the human deoxyhypusine hydroxylase from HeLa cells using a novel enzyme assay

Hypusine synthesis in the eukaryotic initiation factor 5A is a unique two-step posttranslational modification. After deoxyhypusine is generated by the deoxyhypusine synthase, the deoxyhypusine hydroxylase (EC 1.14.99.29) catalyzes the formation of mature hypusine. A rapid assay for monitoring the deoxyhypusine hydroxylase activity was established, employing the oxidative cleavage of the hypusyl residue and subsequent extraction of the generated aldehydes. As metal ion chelators have been reported to inhibit the deoxyhypusine hydroxylase, the mechanism of this inhibition and the effect of transition metal ions on the enzyme activity were investigated. A ferric ion appears to be essential for enzymatic activity, the inhibition of which is entirely attributed to the metal ion bunding capacity of the chelators.     

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.     

Developmental Pattern for Deoxyhypusine Hydroxylase in Rat Brain

Deoxyhypusine hydroxylase catalyzes the formation of hypusine from deoxyhypusine in a precursor form of eukaryotic initiation factor 4D (eIF-4D). The enzymatic activity was examined in mammalian brain homogenates and the results were consistent with the existence of deoxyhypusine hydroxylase levels comparable to those occurring in other mammalian tissues. Interspecies differences in the enzyme distribution were quite limited, with the highest specific activity values observed in cow brain (1.82 units/ mg of protein). In the rat the enzyme was found to be unevenly distributed among various brain regions. The parietal cortex contained the highest specific activity (2.1 units/mg of protein). Rat brain deoxyhypusine hydroxylase was mainly present in the postmicrosomal supernatant (81% of the total activity). The highest specific activity (3 units/mg of protein) was observed in the rat brain during the first few days of life. Thereafter the activity started to decline, and continued to do so for 15 days, remaining throughout the rest of life at levels of less than one-half that of newborn.     

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.     

Deoxyhypusine hydroxylase from rat testis. Partial purification and characterization

Deoxyhypusine hydroxylase, the enzyme that catalyzes the formation of hypusine from deoxyhypusine in eukaryotic initiation factor 4D, has been partially purified from rat testis. The partially purified enzyme requires only the addition of certain sulfhydryl compounds for catalytic activity, dithiothreitol being the most effective. Its lack of dependency on the alpha-keto acid-dependent dioxygenase cofactors, Fe2+, alpha-ketoglutarate, and ascorbic acid, its failure to decarboxylate stoichiometrically alpha-ketoglutarate with deoxyhypusine hydroxylation, and its strong and specific inhibition by Fe2+ all suggest a catalytic mechanism of this enzyme unlike that of the prolyl and lysyl hydroxylases.     

The biosynthesis of hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). Alignment of the butylamine segment and source of the secondary amino nitrogen

The unusual amino acid hypusine is produced in a single protein of mammalian cells by a novel posttranslational event in which a lysine residue is conjugated with the four-carbon moiety from the polyamine spermidine to form an intermediate deoxyhypusine, and in which this intermediate is subsequently hydroxylated. Specifically isotopically labeled precursors of hypusine were used to identify the biosynthetic origin of some of the atoms of hypusine and thus to provide further insight into the mechanism of this in vivo chemical modification reaction. Radiolabel from [1,4-3H] putrescine, [1,8-3H]spermidine, and [5-3H]spermidine entered hypusine during growth of Chinese hamster ovary cells. The occurrence of this label at positions 1 and 4, at position 4, and at position 1, respectively, in the 4-amino-2-hydroxybutyl portion of hypusine revealed an alignment of atoms identical to that in the butylamine segment of spermidine. Growth of cells with [epsilon-15N]lysine as the source of lysine yielded hypusine enriched in 15N, whereas only isotope-free hypusine during growth by [4-15N]spermidine. These was found in cells whose spermidine was replaced during growth by [4-15N]spermidine. These findings are in accordance with a proposal that the first phase of hypusine biosynthesis, the production of intermediate deoxyhypusine, occurs through transfer of the butylamine moiety from spermidine to the epsilon-amino nitrogen of protein-bound lysine. The technique of thermospray high-performance liquid chromatography/mass spectrometry provided positive identification of 15N in hypusine through final separation and on-column direct analysis of this amino acid. Methods of preparation are given for spermidine of high specific radioactivity, labeled specifically at position 5 with 3H, and for spermidine with 15N at the 4-position.     

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 identification of an eukaryotic initiation factor 4D precursor in spermidine-depleted Chinese hamster ovary cells

When Chinese hamster ovary cells are incubated with [terminal methylenes-3H]spermidine, radioactivity is incorporated into a single cellular protein, eukaryotic initiation factor 4D (eIF-4D), through posttranslational synthesis of the amino acid hypusine (N epsilon-(4-amino-2-hydroxybuyly)lysine). The effect of spermidine depletion on this protein modification reaction was studied by high resolution two-dimensional gel electrophoresis. Factor eIF-4D containing both [3H]lysine and [3H]hypusine was detected as one of the major labeled cellular proteins on the fluorographic map of the proteins from Chinese hamster ovary cells that had been incubated with [3H]lysine. When these cells were depleted of spermidine by the use of DL-alpha-difluoromethylornithine before addition of [3H]lysine, no radiolabeling of this mature eIF-4D (hypusine form, Mr approximately 18,000; pI approximately 5.3) occurred. Instead, a new radiolabeled protein (Mr 18,000; pI 5.1) that contained [3H]lysine but no [3H]hypusine or [3H]deoxyhypusine was seen. This protein was identified as an eIF-4D precursor by comparison of the two-dimensional map of its tryptic peptides with that of the tryptic peptides from [3H]lysine-labeled eIF-4D. Further comparisons also suggest that additional post-translational modification processes are involved in the biogenesis of eIF-4D.     

Specificity of the deoxyhypusine hydroxylase-eukaryotic translation initiation factor (eIF5A) interaction: identification of amino acid residues of the enzyme required for binding of its substrate, deoxyhypusine-containing eIF5A

Deoxyhypusine hydroxylase (DOHH) is a novel metalloenzyme that catalyzes the final step of the post-translational synthesis of hypusine (Nepsilon-(4-amino-2-hydroxybutyl)lysine) in the eukaryotic translation initiation factor 5A (eIF5A). Hypusine synthesis is unique in that it occurs in only one protein, denoting the strict specificity of the modification enzymes toward the substrate protein. The specificity of the interaction between eIF5A and DOHH was investigated using human eIF5A (eIF5A-1 isoform) and human recombinant DOHH. DOHH displayed a strong preference for binding the deoxyhypusine-containing form of eIF5A, over the eIF5A precursor or the hypusine-containing eIF5A, indicating a role for the deoxyhypusine residue in binding. In addition to the deoxyhypusine residue, a large portion of the eIF5A polypeptide (>20-90 amino acids) is required for effective modification by DOHH. We have identified the amino acid residues of DOHH that are critical for substrate binding by alanine substitution of 36 conserved amino acid residues. Of these, alanine substitution at Glu57, Glu90, Glu208, Glu241, Gly63, or Gly214 caused a severe impairment in eIF5A(Dhp) binding, with a complete loss of binding and activity in the E57A and E208A mutant enzymes. Only aspartate substitution mutants, E57D or E208D, retained partial activity and substrate binding, whereas alanine, glutamine, or asparagine mutants did not. These findings support a proposed model of DOHH-eIF5A binding in which the amino group(s) of the deoxyhypusine side chain of the substrate is primarily anchored by gamma-carboxyl groups of Glu57 and Glu208 at the DOHH active site.     

Mutational analyses of human eIF5A-1--identification of amino acid residues critical for eIF5A activity and hypusine modification

The eukaryotic translation initiation factor 5A (eIF5A) is the only protein that contains hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine], which is required for its activity. Hypusine is formed by post-translational modification of one specific lysine (Lys50 for human eIF5A) by deoxyhypusine synthase and deoxyhypusine hydroxylase. To investigate the features of eIF5A required for its activity, we generated 49 mutations in human eIF5A-1, with a single amino acid substitution at the highly conserved residues or with N-terminal or C-terminal truncations, and tested mutant proteins in complementing the growth of a Saccharomyces cerevisiae eIF5A null strain. Growth-supporting activity was abolished in only a few mutant eIF5As (K47D, G49A, K50A, K50D, K50I, K50R, G52A and K55A), with substitutions at or near the hypusine modification site or with truncation of 21 amino acids from either the N-terminus or C-terminus. The inactivity of the Lys50 substitution proteins is obviously due to lack of deoxyhypusine modification. In contrast, K47D and G49A were effective substrates for deoxyhypusine synthase, yet failed to support growth, suggesting critical roles of Lys47 and Gly49 in eIF5A activity, possibly in its interaction with effector(s). By use of a UBHY-R strain harboring genetically engineered unstable eIF5A, we present evidence for the primary function of eIF5A in protein synthesis. When selected eIF5A mutant proteins were tested for their activity in protein synthesis, a close correlation was observed between their ability to enhance protein synthesis and growth, lending further support for a central role of eIF5A in translation.     

A rapid and robust assay for the determination of the amino acid hypusine as a possible biomarker for a high-throughput screening of antimalarials and for the diagnosis and therapy of different diseases

Eukaryotic initiation factor 5A (eIF5A) has recently been identified as a biomarker of prognostic significance and therapeutic potential for the treatment in hepatocellular carcinoma. This prompted us to establish a rapid and robust assay to determine deoxyhypusine and hypusine formed with the purified enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) from Plasmodium to develop a rapid screening assay for antimalarial drugs. The peptide hydrolysate obtained from hypusinylated eIF5A was analyzed by ultra performance liquid chromatography (UPLC) with retention times for deoxyhypusine of 7.44 min and for hypusine of 7.30 min, respectively. The limit of detection for both compounds was 0.144 ng/μl. Determination of the specific activity of Plasmodium DOHH resulted in a twofold higher specific activity than its human counterpart. Following the iron-complexing strategy of the ferrous iron which is present in the active site of Plasmodium DOHH, a series of iron chelating compounds was tested. 2,2′-Dipyridyl and mimosine abolished DOHH activity completely while 4-oxo-piperidine-carboxylates i.e. the nitrophenylether JK8-2 and EHW 437, the oxime ether of the piperidine aldehyde, showed no inhibition although they were highly active in in vitro cultures of Plasmodium and in vivo in a rodent mouse model. The method allows a high-throughput screening (HPTS) of antimalarial drugs and the evaluation of eIF5A as a biomarker.     

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.     

Characterization and reconstitution of a cell free system for NAD(+)-dependent deoxyhypusine formation on the 18 kDa eIF-4D precursor

Deoxyhypusine formation on the 18 kDa eIF-4D precursor is due to a covalent linkage between a lysine residue of the protein and the aminobutyl moiety derived from spermidine. The deoxyhypusine is then hydroxylated to form hypusine. This post-translational modification represents one of the most specific spermidine-dependent biochemical events in eukaryotic cells. Deoxyhypusine formation can be performed in vitro at pH 9.5 and is greatly stimulated by NAD+. Using the labeling of the 18 kDa protein by [3H]spermidine as an assay for deoxyhypusine formation, we found that (i) significant deoxyhypusine formation can be demonstrated in vitro at pH 7.2 only if NAD+ is present, (ii) deoxyhypusine formation was sensitive to buffer composition; buffers made of basic amino acids and Tris were inhibitory, (iii) sulfhydryl reagents and metal ions such as Cu2+ and Fe3+ were potent inhibitors of deoxyhypusine formation and (iv) the 18 kDa protein substrate was heat-stable. The in vitro activity of deoxyhypusine formation, which depends on the presence of both enzyme and protein substrate, can be separated from the product, eIF-4D, by a one-step Cibacron blue dye affinity column. Taking advantage of this finding, we have developed a simple procedure, based on the use of Cibacron blue dye, for partially purifying both the deoxyhypusine-forming enzyme and the 18 kDa protein substrate. When the partially purified enzyme and protein substrate were mixed in the presence of 1 mM NAD+ and [3H]spermidine, the 18 kDa protein was radiolabeled, no labeling could be detected if any one component was absent. Using partially purified enzyme, we have also determined the half-life of the protein substrate in alpha-difluoromethyl ornithine (DFMO)-treated NB-15 cells and found it to be longer than 10 h.     

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.