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 mammalian hypusine-containing protein, eukaryotic initiation factor 4D. Structural homology of this protein from several species

A single cellular protein of Mr approximately 18,000 and pI near 5.1, recently identified as eukaryotic translation initiation factor eIF-4D, contains the unusual amino acid hypusine [N epsilon-(4-amino--2-hydroxybutyl)lysine] formed post-translationally from lysine with a structural contribution from the polyamine spermidine. When the 3H-labeled hypusine-containing protein isolated from Chinese hamster ovary (CHO) cells that were grown with radioactive polyamine is digested with trypsin and the digest is subjected to two-dimensional separation, a single radioactive peptide is seen. A labeled peptide that occupies this same position is found in a digest of the [3H]hypusine protein from human lymphocytes and the single hypusine-containing tryptic peptide from purified rabbit reticulocyte eIF-4D also moves to this identical position. Stepwise Edman degradation of the tryptic digest of CHO cell hypusine-protein releases the radioactivity as a single peak in accordance with our earlier evidence for a single hypusine residue per molecule of eIF-4D. The similar patterns of radioactive peptides obtained from tryptic digests of radioiodinated eIF-4D from CHO cells, human lymphocytes, and rabbit reticulocytes suggest a highly conserved primary structure for this protein.     

Two isoforms of eIF-5A in chick embryo. Isolation, activity, and comparison of sequences of the hypusine-containing proteins.

Eukaryotic translation initiation factor 5A (eIF-5A) (older terminology, eIF-4D) is unique in that it contains the unusual amino acid hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). Hypusine is formed by a post-translational event in which a specific lysine residue is modified by a structural contribution from spermidine. Metabolic labeling of chick embryo fibroblasts with [3H]spermidine or [3H]lysine gives rise to two distinct proteins, designated I (approximately 20 kDa and pI 5.6) and II (approximately 18 kDa and pI 5.35), that contain [3H]hypusine. Upon incubation with [3H]lysine the labeling of the two proteins followed a similar time course and showed approximately the same ratio over the 6-h incubation period. [3H]Hypusine-containing proteins from cells which had been cultured with [3H]spermidine were employed as tracers for isolation of hypusine-containing proteins from whole chick embryos. Four such proteins were obtained. Two of these proteins, I and II, correspond to the two native proteins synthesized in chick embryo fibroblasts; the other two forms, Ia and IIa, displayed properties suggesting that they were derived from the native proteins, I and II, respectively, during purification. The amino acid compositions and the tryptic peptide maps of the 20-kDa protein (I) and the 18 kDa protein (II) suggest that they are closely related but distinct proteins. In fact, amino acid sequence analysis of the two major proteins revealed differences in the polypeptide backbone of the two proteins. In spite of structural differences, the two native forms (I and II), as well as the two altered forms (Ia and IIa), were effective in stimulating methionyl-puromycin synthesis, providing evidence that they are indeed functional isoforms of eIF-5A.     

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.     

The essential role of hypusine in eukaryotic translation initiation factor 4D (eIF-4D). Purification of eIF-4D and its precursors and comparison of their activities

Eukaryotic translation initiation factor 4D (eIF-4D) is the only protein known to contain the amino acid, hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine]. This unusual amino acid is formed post-translationally by modification of a single specific lysine residue in an eIF-4D precursor protein. Two separate eIF-4D precursors, each of which contains a lysine residue in place of the hypusine residue and each of which thereby serves as a protein substrate for the hypusine modification, were purified from DL-2-difluoromethylornithine-treated Chinese hamster ovary cells by means of a five-step procedure. These two precursors termed PI and PII both have apparent molecular masses of approximately 17 kDa, indistinguishable from that of eIF-4D, but exhibit more acidic isoelectric points (5.1 and 5.25 for PI and PII, respectively, compared with 5.37 for eIF-4D). These physical characteristics, together with other properties, indicate that eIF-4D differs from PII only in possessing the hypusine residue in place of a lysine residue, whereas an additional structural difference exists between PI and eIF-4D. eIF-4D from CHO cells provides a significant enhancement of methionyl-puromycin synthesis, a model assay for translation initiation. Neither PI nor PII stimulates this in vitro system. These findings are the first direct evidence that hypusine is essential for the biological activity of eIF-4D.     

cDNA and derived amino acid sequence of the hypusine containing protein from Dictyostelium discoideum

The eukaryotic translation initiation factor eIF-4D is the only protein known to contain the unusual amino acid hypusine, a posttranslationally modified lysine. For the production of monoclonal antibodies the hypusine-containing protein (HP) was isolated from Dictyostelium discoideum. Using these monoclonal antibodies, a full-length cDNA clone was isolated from a lambda gt11 library. The D. discoideum HP consists of 169 amino acids and has a molecular mass of 18.3 kDa. It is encoded by a single gene. Tryptic and cyanogen bromide peptides were prepared from the purified protein and sequenced. The hypusine residue is located at amino acid position 65 of the HP. The corresponding mRNA of approx. 0.6 kb is present throughout the life cycle of D. discoideum.     

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.     

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.     

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.     

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.     

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.     

Sequence determination and cDNA cloning of eukaryotic initiation factor 4D, the hypusine-containing protein

Protein synthesis initiation factor 4D (eIF-4D) from mammalian cells contains the post-translationally modified lysine derivative hypusine. A highly purified preparation of the protein from rabbit reticulocytes was subjected to chemical and enzymatic cleavage, and a large number of overlapping peptides were resolved by high performance liquid chromatography and sequenced. Two mixed 14-base DNA probes were synthesized based on suitable amino acid sequences and were used to screen a human cDNA library in lambda gt11. A cDNA insert containing eIF-4D encoding sequences was identified and a 558-base pair EcoRI-PstI fragment was sequenced. Northern blot hybridization of HeLa cell RNA shows a single size class (1.2 kilobase) of mRNA. The DNA encodes a protein comprising 154 residues with a mass of 16,703 daltons. Human eIF-4D matches all of the rabbit peptides sequenced, extending from residue 9 to 154 except for Cys-129 which is Ser in the rabbit protein. The residue modified to hypusine is identified as Lys-50 and the amino terminus is blocked. eIF-4D possesses rather little secondary structure in the amino-terminal two-thirds of the protein, but the carboxyl-terminal third is rich in alpha helices.     

Protein synthesis initiation factor eIF-4D. Functional comparison of native and unhypusinated forms of the protein

Protein synthesis initiation factor eIF-4D is a relatively abundant protein in mammalian cells and possesses a unique amino acid residue, hypusine. The role of the hypusine modification in eIF-4D function was addressed by studying the function of eIF-4D variants lacking hypusine. The cloned human cDNA encoding eIF-4D was overexpressed in Escherichia coli and a precursor form lacking hypusine was purified. This protein fails to stimulate methionyl-puromycin synthesis in vitro, nor does it significantly inhibit the action of native eIF-4D. Mammalian expression vectors were constructed with the wild-type cDNA and a mutant form in which the codon for lysine-50 (the residue hypusinated) was altered by site-directed mutagenesis to that for arginine. Transient co-transfection of COS-1 cells with the eIF-4D vector and a vector expressing dihydrofolate reductase led to strong synthesis of both eIF-4D and dihydrofolate reductase. This indicates that normal cellular levels of eIF-4D are saturating in these cells and that excess levels of eIF-4D are not detrimental. Cotransfection with the eIF-4D arginine variant caused no effect on dihydrofolate reductase synthesis, in agreement with the in vitro experiments. The inability of the unhypusinated eIF-4D variants to stimulate methionyl-puromycin synthesis in vitro and to affect protein synthesis in vivo strongly suggests that the hypusine modification is required for eIF-4D activity and for its interaction with the 80 S initiation complex in protein synthesis.     

结构生物学研究中的eIF-5A的表达与纯化

真核细胞翻译官始因子eIF-5A（eukaryotic initiation factor 5A）是迄今发现的惟一含有特殊氨基酸hypusine残基的蛋白质,其具体生物学功能仍不明确。为了推进对其功能的研究,拟从结构生物学入手,对其结构进行核磁共振（NMR）结构解析。利用GST融合蛋白原核表达系统,将eIF-5A进行原核表达,经过优化表达与纯化条件,得到了高产率与高纯度的可溶性eIF-5A用以进行NMR测试：经过!1H-^15N HSQC NMR实验,发现其适合应用NMR方法进行结构解析,从而为溶液中eIF-5A三维构象的研究奠定了基础.     

Cleavage of spermidine as the first step in deoxyhypusine synthesis. The role of NAD

The biosynthesis of deoxyhypusine (N-(4-aminobutyl)lysine) occurs by the transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in a precursor of eukaryotic translation initiation factor 4D (eIF-4D). Deoxyhypusine synthase, the enzyme that catalyzes this reaction, was purified approximately 700-fold from rat testis. The Km values for the substrates, spermidine, the eIF-4-D precursor protein, and NAD+, were estimated as approximately 1, 0.08, and 30 microM, respectively. After incubation of partially purified enzyme with [1,8-3H]spermidine, NAD+, and the eIF-4D precursor, equal amounts of radioactivity were found in free 1,3-diaminopropane and in protein-bound deoxyhypusine. However, when the protein substrate (eIF-4D precursor) was omitted, radioactivity was found in 1,3-diaminopropane and in delta 1-pyrroline in nearly equal quantities, providing evidence that the cleavage of spermidine occurs, albeit at a slower rate, in the absence of the eIF-4D precursor. That NAD+, which is required for this reaction, functions as the hydrogen acceptor was demonstrated by the fact that radioactivity from spermidine labeled with 3H at position 5 is found in NADH as well as in delta 1-pyrroline. Transfer of this hydrogen from spermidine to the re face of the nicotinamide ring of NAD+, as determined by the use of dehydrogenases of known stereospecificity, defines the first step of deoxyhypusine synthesis as a pro-R, or A, stereospecific dehydrogenation. Based on these findings, an enzyme mechanism involving imine intermediate formation is proposed.     

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.     

Eukaryotic initiation factor 4D. Purification from human red blood cells and the sequence of amino acids around its single hypusine residue

Eukaryotic initiation factor 4D (eIF-4D) was purified from human red blood cells by a simple 5-step procedure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the preparations of eIF-4D were composed of variable amounts of two closely migrating forms of the factor, each of which contained a single residue of the unique amino acid hypusine. The structural similarity of the two forms of human eIF-4D was evidenced by the indistinguishable patterns of radioactivity on peptide maps of tryptic digests prepared from radioiodinated samples. A peptide containing the single hypusine residue was readily isolated from a tryptic digest of human eIF-4D by virtue of its high positive charge and hydrophilic character. Amino acid sequence determination on this peptide revealed the following primary structure around hypusine: Thr-Gly-hypusine-His-Gly-His-Ala-Lys.     

Eukaryotic initiation factor 4D, the hypusine-containing protein, is conserved among eukaryotes

When mammalian cells are grown in medium containing [3H]spermidine, a single major tritiated protein identical to eukaryotic initiation factor 4D becomes labeled. This protein contains 1 residue/molecule of tritiated hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine), a rare amino acid which has been found in no other protein. In order to investigate the conservation of this protein, we examined two nonmammalian eukaryotes, the yeast Saccharomyces cerevisiae and the insect Drosophila melanogaster, and the eubacterial prokaryote Escherichia coli for the presence of the hypusine-containing protein. When the eukaryotic cells were grown in the presence of [3H]spermidine, electrophoretic analysis revealed a single labeled protein. In each case, the apparent molecular weight was near 18,000 and the relative pI was approximately 5.2, similar to the hypusine-containing protein of mammals. Amino acid analysis confirmed the presence of tritiated hypusine in each case, and silver staining of two-dimensional polyacrylamide gels demonstrated that, in yeast and fruit flies as in mammals, the protein is relatively abundant. In the eubacterium E. coli, one tritiated protein was predominant, but its molecular weight was 24,000 and we found no evidence that it contained tritiated hypusine. We found no evidence for the existence of the hypusine-containing protein in the archaebacterium Methanococcus voltae. These data suggest that the hypusine-containing protein is conserved among eukaryotes.     

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.