In previous studies we have identified eIF5A as the only cellular protein that contains an unusual amino acid, hypusine N-epsilon-(4-amino-2-hydroxybutyl)lysine, and established that hypusine biosynthesis occurs by two sequential enzymatic reactions: i) deoxyhypusine synthesis and ii) deoxyhypusine hydroxylation. We have cloned and characterized the structural and catalytic properties of the two enzymes of the hypusine pathway, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). We and others have demonstrated that hypusine modification is essential for the activity of eIF-5A and for mammalian cell proliferation. Previously, we and others reported biochemical evidence for acetylation of eIF5A at Lys47 and/or Lys68 and its negative regulation by acetylation at Lys 47. We have also obtained evidence for acetylation of eIF5A at the hypusine residue by the polyamine-acetylating enzyme, spermidine/spermine N1-acetyltransferase (SSAT1), and for the inactivation of eIF5A by the SSAT1-mediated hypusine acetylation. In the course of study of molecular interaction between eIF5A and SSAT1, we have observed a strong suppression of expression of exgogenous gene expression by SSAT1. This suppression was dependent on the SSAT1 activity and was limited to exogenous expression of any transiently transfected genes. The process does not apprear to involve activation of proteases or nucleases and does not correlate with depletion of overall cellular polyamine pool, suggesting new fuction and targets for SSAT1. No effects by exogenous SSAT1 were observed on any endogenous gene expression, suggesting differences between the molecular mechanisms of endogenous vs exogenous gene expression pathways. eIF5A is a putative translation initiation factor that stimulates methionyl-puromycin synthesis. However, recent genetic studies in S. cerevisiae and D. melanogaster implicate a role for eIF5A at the elongation step of translation. Hypusine modification is required for the activity of eIF5A in translation and for its binding to the ribosome. By using an inhibitor of polyamine biosynthesis, difluoro-methylornithine (DFMO), we have obtained evidence suggesting that polyamines regulate transaltion initiation through their effects on phosphorylation of eIF2 alpha and eIF4E-BP. Our data from cells depleted of eIF5A by the eIF5A SiRNA and by an inhibitor of hypusine modification suggest a role for eIF5A in the elongation step of translation in mammalian cells. To investigate the physiological function of eIF5A isoform 1 and deoxyhypusine synthase, we performed their gene targeting in mice. We used the ES cell lines, RRE174 (Eif5a1 +/-) and RRM039 (Dhps +/-) which have one allele of the Eif5a1 or the Dhps gene disrupted by the gene trap method. After confirmation of disruption of each gene by PCR of genomic DNA isolated from these ES cells, the RRE174 and RRM039 cells were injected into blastocysts of C57 and the injected blastocysts were implanted into pseudo-pregnant C57 female mice. Male chimeras of each clone were mated with C57 females to produce agouti pups. The gene-targeted heterozygous agouti mice (Eif5a1+/-, or Dhps+/-) appeared to be normal and did not show any gross growth defects or phenotypes. The heterozygous agouti male and female mice were crossed and the pups born from the heterozygous intercrosses were genotyped to determine if homozygous disruption of Eif5a1 or Dhps is lethal or not. Of 65 pups born from over 10 heterozygous intercrosses (Eif5a1 +/-), no pups were born with Eif5a1 homozygous KO genotype. Thus, Eif5a1 gene disruption must cause embryonic lethality in mice. The Eif5a1 -/- homozygous embryo was identified on the blastocyst stage (E3.5), but not at later stage, indicating that Eif5a1 -/- embryo is viable up to 3.5 days. The phenotype of Dhps homozygous gene disruption in mice was also embryonic lethal. Of 52 pups born from 11 heterozygous agoouti intercrosses, no pups with homozygous Dhps disruption were found. The Dhps -/- homozygous embryo was identified on the blastocyst stage (E3.5), but not at later stage. These results indicate that both eIF5A-1 and DHS play an essential role for early embryonic development in mice.
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