The Post-Translational Synthesis of a Polyamine-Derived Amino Acid, Hypusine, in the Eukaryotic Translation Initiation Factor 5A (eIF5A)

Polyamines can be covalently incorporated into proteins through the transglutaminase reaction and by hypusine biosynthesis. In the latter, the polyamine, spermidine, is utilized for the post-translational modification of a single cellular protein, the precursor of eukaryotic translation initiation factor 5A (eIF5A), to form a unique amino acid, hypusine [Ne-(4-amino-2-hydroxybutyl)lysine] (1–4). Hypusine was originally discovered and identified by Shiba et al. and was named “hypusine” based on its structure, which consists of two moieties, hydroxyputrescine and lysine (5). In the course of a study aimed at the identification of a cellular protein substrate of trans-glutaminase, we discovered that hypusine is, in fact, formed from spermidine in two enzymatic steps (6) (Fig. 1): in the first step, deoxyhypusine synthase [EC 2.5.1.46] catalyzes the transfer of the 4-amino butyl moiety of spermidine to the e-amino group of one specific lysine residue (Lys50 for the human protein) of the eIF5A precursor to form a deoxyhypusine residue (7–9) This intermediate is subsequently hydroxylated by deoxyhypusine hydroxylase [EC 1.14.99.29] to complete hypusine synthesis and eIF5A maturation (10). The biosynthesis of hypusine defines a specific and critical function of polyamines in cell proliferation (2, 3, 11–13). Fig. 1 Scheme of hypusine biosynthesis in eIF5A eIF5A is a small, acidic protein that is highly conserved throughout eukaryotes. Sequence conservation is especially high around the hypusine residue, underscoring the importance of this unusual protein modification throughout eukaryotic evolution. The early observation of a correlation between hypusine synthesis and cell growth suggested an important role for hypusine in cell proliferation (14–16). Inhibitors of deoxyhypusine synthase and deoxyhypusine hydroxylase cause growth arrest in various mammalian cells (17, 18). Furthermore, the cytostasis observed in spermidine-deprived cells after treatment with an inhibitor of S-adenosylmethionine decarboxylase was attributed to depletion of hypusine-containing eIF5A (11). Direct evidence for the essential nature of eIF5A and its modification derives from gene inactivation studies in Saccharomyces cerevisiae. Disruption of the eIF5A genes (TIF51A and TIF51B) (19, 20), or of the deoxyhypusine synthase gene (21, 22), produces a lethal phenotype, indicating the importance of the deoxyhypusine modification in the viability of eukaryotic cells. The second step, deoxyhypusine hydroxylation, was also presumed to be essential, based on cell growth inhibition by inhibitors of this enzyme. Recent cloning and identification of the deoxyhypusine hydroxylase gene (23) provide new insights into the evolutionary progression of eIF5A protein and its modification enzymes. Although eIF5A is intimately involved in eukaryotic cell proliferation, the true physiological function of this essential factor has yet to be elucidated. eIF5A stimulates methionyl-puromycin synthesis (24), in a model assay for the first peptide bond formation. eIF5A may be a bimodular protein interacting with both RNA and proteins, and is presumed to have an important role in the translation machinery. However, a rapid depletion of genetically engineered unstable eIF5A in S. cerevisiae resulted in only a modest decrease in total protein synthesis (25). This finding led to a proposal that eIF5A is an initiation factor specific for a subset of mRNA’s (25, 26). Additional proposed functions of eIF5A include: a cellular cofactor of HIV-1 REV (27), a factor involved in nuclear export (28) and mRNA turnover (29, 30). Recent studies with temperature-sensitive mutants and their suppressors point to a role of eIF5A in cell wall integrity, and actin polarity (31). It is not clear whether the various observed phenotypes are direct or indirect consequence of eIF5A depletion or dysfunction and how the various effects are interrelated. It is possible that eIF5A is a multifunctional protein involved in several critical cellular processes.