The Post-translational Synthesis of Hypusine In eIF5A

Dental &Craniofacial Research

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Abstract

We have identified eIF5A as the only cellular protein that contains an unusual amino acid, hypusine [Nepsilon- (4-amino-2-hydroxybutyl)lysine], and have established that hypusine biosynthesis occurs posttranslationally by two sequential enzymatic reactions. In the first step deoxyhypusine synthase catalyzes the transfer of the butylamine moiety of the polyamine spermidine to a specific lysine residue in the eIF-5A precursor protein to form an intermediate, deoxyhypusine residue. In the latter step, this intermediate is converted to hypusine by a metalloenzyme deoxyhypusine hydroxylase. Hypusine is essential for the activity of eIF-5A and for eukaryotic cell proliferation. Inhibitors of hypusine biosynthesis exert strong anti-proliferative effects in mammalian cells. Ciclopirox, an anti-fungal drug and inhibitor of deoxyhypusine hydroxylase exhibits anti-angiogenic activity and inhibits growth of various human cancer cell lines including the cervical cancer line SiHa, by causing a cell cycle arrest at G1/S boundary. We conducted structure/function studies of eIF5A, using truncated forms of eIF5A and mutant forms with single amino acid substitutions . The results indicate that 10 amino acids either from the amino terminal or the carboxyl terminal are indispensible for eIF5A activity. The amino acid residues thus far identified to be critical for eIF5A function are Lys47, Gly49, Lys50, His51, Gly52, Lys55, Leu91, Leu101 and Glu143. We compared the expression and function of the two human eIF5A genes. Complementation studies in yeast indicate that both genes encode bona fide eIF5A proteins with similar basic cellular function. Although the two human eIF5A isoforms are highly similar in sequence (84% identity) they exhibit distinct immunoreactivity and kinetic values as substrates of deoxyhypusine synthase. Furthermore, the two eIF5A genes are differentially regulated. Whereas eIF5A-1 is highly expressed in all human cells, eIF5A-2 protein is not normally expressed. eIF5A-2 expression is limited to some specific tissues or cancer cells such as UACC1598 and SW480 and it has been suggested as an oncogene. Northern and RTPCR experiments suggest that the low expression of eIF5A-2 isoform is largely due to the poor translatability of eIF5A-2 mRNA and that its 3' UTR is responsible for the inefficient translation.