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. The inhibitors of deoxyhypusine synthase and deoxyhypusine hydroxylase exert strong antiproliferative effects in mammalian cells. We have tested the effects of inhibitors of deoxyhypusine hydroxylase on human vein endothelial cell (HUVEC) proliferation and angiogenesis. These compounds inhibited deoxyhypusine hydroxylase and proline hydroxylase, and caused cell cycle arrest in G1. Of the five metal chelating inhibitors i.e. mimosine, 2,2'-dipyridyl, deferiprone, deferoxamine and ciclopirox, the antifungal drug ciclopirox was the most effective in the inhibition of the two protein hydroxylases, HUVEC proliferation and angiogenesis in two model assays. Furthermore, this compound exerts strong antiproliferative effects on a panel of human cancer cell lines. These findings suggest that ciclopirox is a valuable candidate for clinical trials in the treatment of solid tumors. We conducted structure/function studies of eIF5A, using truncated forms of eIF5A and mutant enzymes. 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, Lys50, His51, Pro82 and Pro114. 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. However, the two genes are differentially regulated and in most human cells, one isoform, eIF5AI is expressed: Only in two human cancer lines, UACC1598 and SW480, both eIF5AI and eIF5AII are detected. Northern and RTPCR experiments suggest that the poor translatability of EIF5A2 mRNA is due to its 3' UTR.