Edith C. Wolff

Is hypusine essential for eukaryotic cell proliferation

Hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine] occurs in all eukaryotes at one residue in a highly conserved protein, the putative eukaryotic translation initiation factor 5A (eIF-5A, old terminology eIF-4D). This unusual amino acid is produced in a unique posttranslational modification reaction that involves the conjugation of the 4-aminobutyl moiety of the polyamine spermidine to the epsilon-amino group of a specific lysine residue of the eIF-5A precursor protein to form the deoxyhypusine [N epsilon-(4-aminobutyl)lysine] residue and its subsequent hydroxylation. The strict specificity of hypusine synthesis, its derivation from spermidine and its requirement for the activity of eIF-5A and for eukaryotic cell proliferation have raised keen interest in the physiological function of the hypusine-containing protein, eIF-5A.

The antifungal drug ciclopirox inhibits deoxyhypusine and proline hydroxylation, endothelial cell growth and angiogenesis in vitro

The hypusine biosynthetic steps represent novel targets for intervention in cell proliferation. Hypusine is a rare amino acid, formed posttranslationally in one cellular protein, eIF5A, and is essential for cell proliferation. Deoxyhypusine hydroxylase, the metalloenzyme catalyzing the final step in hypusine biosynthesis, and prolyl 4‐hydroxylase, a non‐heme iron enzyme critical for collagen processing, can be inhibited by small chelating molecules that target their essential metal atom. We examined the effects of 5 compounds (ciclopirox, deferiprone, deferoxamine, mimosine and 2,2′‐dipyridyl) on these protein hydroxylases in HUVECs, on cell proliferation and on angiogenesis using 2 model assays: tube‐like vessel formation on Matrigel and the chick aortic arch sprouting assay. These compounds inhibited cellular deoxyhypusine hydroxylase in a concentration‐dependent manner, but their efficacy varied widely in the following order: ciclopirox→ deferoxamine→2,2′‐dipyridyl→deferiprone→mimosine (IC50 5–200 μM). Inhibition of DNA synthesis, following the same order (IC50 10–450 μM), correlated with G1 arrest of the cell cycle. These compounds also inhibited proline hydroxylation and maturation of collagen in HUVECs and caused inhibition of angiogenesis in vitro. Of the compounds tested, ciclopirox was by far the most effective inhibitor of HUVEC proliferation and angiogenesis. The strong antiangiogenic activity of this readily available antifungal drug along with its antiproliferative effects suggests a new potential application for ciclopirox in the treatment of solid tumors. Published 2002 Wiley‐Liss, Inc.

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

Summary.A naturally occurring unusual amino acid, hypusine [Nɛ-(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.

Cloning and Expression of Human Deoxyhypusine Synthase cDNA STRUCTURE-FUNCTION STUDIES WITH THE RECOMBINANT ENZYME AND MUTANT PROTEINS

Deoxyhypusine synthase catalyzes the first step in the post-translational formation of hypusine (N-(4-amino-2-hydroxybutyl)lysine). cDNA clones encoding deoxyhypusine synthase were isolated from a human HeLa cell library. Full-length cDNA clones encoding a 369-amino acid protein (calculated molecular mass of 40,970 Da) and a shorter cDNA clone that would potentially encode a protein with an internal deletion of 56 amino acids (Asp-Ser) were isolated. The deduced amino acid sequence of the human enzyme shows a high degree of identity to that of yeast deoxyhypusine synthase and to the known sequences of tryptic peptides from the rat and Neurospora crassa enzymes. The recombinant enzyme formed upon expression in Escherichia coli effectively catalyzed deoxyhypusine synthesis. Variant human recombinant proteins with (i) a truncation of 48 or 97 NH-terminal amino acids, (ii) a truncation of 39 COOH-terminal amino acids, or (iii) an internal deletion (Asp-Ser) were inactive. A chimeric protein consisting of the complete human sequence and 16 amino acids of the yeast sequence (Gln-Asn, not present in the human enzyme) inserted between Glu and Gln exhibited moderate activity.

Differential expression of eIF5A-1 and eIF5A-2 in human cancer cells

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the unusual amino acid hypusine [Nε‐(4‐amino‐2‐hydroxybutyl)lysine]. Vertebrates carry two genes that encode two eIF5A isoforms, eIF5A‐1 and eIF5A‐2, which, in humans, are 84% identical. eIF5A‐1 mRNA (1.3 kb) and protein (18 kDa) are constitutively expressed in human cells. In contrast, expression of eIF5A‐2 mRNA (0.7–5.6 kb) and eIF5A‐2 protein (20 kDa) varies widely. Whereas eIF5A‐2 mRNA was demonstrable in most cells, eIF5A‐2 protein was detectable only in the colorectal and ovarian cancer‐derived cell lines SW‐480 and UACC‐1598, which showed high overexpression of eIF5A‐2 mRNA. Multiple forms of eIF5A‐2 mRNA (5.6, 3.8, 1.6 and 0.7 kb) were identified as the products of one gene with various lengths of 3′‐UTR, resulting from the use of different polyadenylation (AAUAAA) signals. The eIF5A‐1 and eIF5A‐2 precursor proteins were modified comparably in UACC‐1598 cells and both were similarly stable. When eIF5A‐1 and eIF5A‐2 coding sequences were expressed from mammalian vectors in 293T cells, eIF5A‐2 precursor was synthesized at a level comparable to that of eIF5A‐1 precursor, indicating that the elements causing inefficient translation of eIF5A‐2 mRNA reside outside of the open reading frame. On sucrose gradient separation of cytoplasmic RNA, only a small portion of total eIF5A‐2 mRNA was associated with the polysomal fraction, compared with a much larger portion of eIF5A‐1 mRNA in the polysomes. These findings suggest that the failure to detect eIF5A‐2 protein even in eIF5A‐2 mRNA positive cells is, at least in part, due to inefficient translation.

Crystal structure of the NAD complex of human deoxyhypusine synthase: an enzyme with a ball-and-chain mechanism for blocking the active site.

BACKGROUND Eukaryotic initiation factor 5A (elF-5A) contains an unusual amino acid, hypusine [N epsilon-(4-aminobutyl-2-hydroxy)lysine]. The first step in the post-translational formation of hypusine is catalysed by the enzyme deoxyhypusine synthase (DHS). The modified version of elF-5A, and DHS, are required for eukaryotic cell proliferation. Knowledge of the three-dimensional structure of this key enzyme should permit the design of specific inhibitors that may be useful as anti-proliferative agents. RESULTS The crystal structure of human DHS with bound NAD cofactor has been determined and refined at 2.2 A resolution. The enzyme is a tetramer of four identical subunits arranged with 222 symmetry; each subunit contains a nucleotide-binding (or Rossmann) fold. The tetramer comprises two tightly associated dimers and contains four active sites, two in each dimer interface. The catalytic portion of each active site is located in one subunit while the NAD-binding site is located in the other. The entrance to the active-site cavity is blocked by a two-turn alpha helix, part of a third subunit, to which it is joined by an extended loop. CONCLUSIONS The active site of DHS is a cavity buried below the surface of the enzyme at the interface between two subunits. In the conformation observed here, the substrate-binding site is inaccessible and we propose that the reaction steps carried out by the enzyme must be accompanied by significant conformational changes, the least of which would be the displacement of the two-turn alpha helix.

eIF5A isoforms and cancer: two brothers for two functions?

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the unusual amino acid hypusine [Nε-(4-amino-2-hydroxybutyl)lysine]. The role of hypusine formation in the eIF5A protein in the regulation of cell proliferation and apoptosis is addressed in the present review. Moreover, vertebrates carry two genes that encode two eIF5A isoforms, eIF5A-1 and eIF5A-2, which, in humans, are 84% identical. However, the biological functions of these two isoforms may be significantly different. In fact, eIF5A-1 is demonstrable in most cells of different histogenesis, whereas eIF5A-2 protein is detectable only in certain human cancer cells or tissues, suggesting its role as a potential oncogene. In this review we focus our attention on the involvement of eIF5A-1 in the triggering of an apoptotic program and in the regulation of cell proliferation. In addition, the potential oncogenic role and prognostic significance of eIF5A-2 in the prediction of the survival of cancer patients is described. eIF5A-1 and/or the eIF5A-2 isoform may serve as a new molecular diagnostic or prognostic marker or as a molecular target for anti-cancer therapy.

DEOXYHYPUSINE HYDROXYLASE IS A Fe(II)-DEPENDENT, HEAT-REPEAT ENZYME: IDENTIFICATION OF AMINO ACID RESIDUES CRITICAL FOR Fe(II) BINDING AND CATALYSIS

Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of hypusine (Nϵ-(4-amino-2-hydroxybutyl)lysine) in eIF5A. DOHH is a HEAT-repeat protein with eight tandem helical hairpins in a symmetrical dyad. It contains two potential iron coordination sites (one on each dyad) composed of two strictly conserved His-Glu motifs. The purified human recombinant DOHH was a mixture of active holoenzyme containing 2 mol of iron/mol of DOHH and inactive metal-free apoenzyme. The two species could be distinguished by their different mobilities upon native gel electrophoresis. The DOHH apoenzyme exhibited markedly reduced levels of iron and activity. DOHH activity could be restored only by the addition of Fe2+ to the apoenzyme but not by other metals including Cd2+,Co2+,Cr2+,Cu2+,Mg2+,Mn2+,Ni2+, and Zn2+. The role of the strictly conserved His-Glu residues was evaluated by site-directed mutagenesis. Substitution of any single amino acid in the four His-Glu motifs with alanine abolished the enzyme activity. Of these eight alanine substitutions, six, including H56A, H89A, E90A, H207A, H240A, and E241A, caused a severe reduction in the iron content. Our results provide strong evidence that Fe(II) is the active-site-bound metal critical for DOHH catalysis and that the strictly conserved His-Glu motifs are essential for iron binding and catalysis. Furthermore, the iron to DOHH stoichiometry and dependence of iron binding on each of the four conserved His-Glu motifs suggest a binuclear iron mediated reaction mechanism, distinct from that of other Fe(II)-dependent protein hydroxylases, such as prolyl 4-hydroxylase or lysyl hydroxylases.

Reversal of the Deoxyhypusine Synthesis Reaction GENERATION OF SPERMIDINE OR HOMOSPERMIDINE FROM DEOXYHYPUSINE BY DEOXYHYPUSINE SYNTHASE

Deoxyhypusine synthase catalyzes the first step in hypusine (Nϵ-(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 μm). 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.

Enzyme-substrate intermediate formation at lysine 329 of human deoxyhypusine synthase.

Deoxyhypusine (N ε-(4-aminobutyl)lysine) is the key intermediate in the posttranslational synthesis of the unique amino acid, hypusine (N ε-(4-amino-2-hydroxybutyl)lysine). Deoxyhypusine synthase catalyzes the formation of deoxyhypusine by conjugation of the butylamine moiety of spermidine to the ε-amino group of one specific lysine residue of the eukaryotic translation initiation factor 5A (eIF-5A) precursor protein. However, in the absence of the eIF-5A precursor, catalysis involves only the NAD-dependent cleavage of spermidine to generate 1,3-diaminopropane and a putative 4-carbon amine intermediate that gives rise to Δ1-pyrroline. We have obtained evidence for a covalent enzyme-substrate intermediate that accumulates in the absence of the eIF-5A precursor. Incubation of human recombinant enzyme with [1,8-3H]spermidine and NAD, followed by reduction with NaBH3CN, resulted in specific radiolabeling of the enzyme. The radioactive component in the reduced enzyme intermediate was identified as deoxyhypusine and was shown to occur at a single locus. The fact that labeled deoxyhypusine was found after treatment with a reducing agent suggests an intermediate with the butylamine moiety derived from spermidine attached through an imine linkage to the ε-amino group of a specific lysine residue of the enzyme. This residue has been identified as lysine 329. Separate experiments showing efficient transfer of labeled butylamine moiety from enzyme intermediate to eIF-5A precursor strongly support a reaction mechanism involving an imine intermediate.