The Post-translational Synthesis of Hypusine In eIF5A

在之前的研究中，我们已经确定了eIF5A是唯一含有一种不寻常的氨基酸，hypusine N-epsilon-（4-氨基-2-羟基丁基）赖氨酸的细胞蛋白，并确定了hypusine的生物合成发生在两个连续的酶促反应中：i)脱氧hypusine合成和ii)脱氧hypusine羟基化。我们克隆并鉴定了hypusine途径的两个酶，脱氧hypusine合成酶（DHS）和脱氧hypusine羟化酶（DOHH）的结构和催化性能。我们和其他人已经证明，hypusine修饰对于eIF-5A的活性和哺乳动物细胞增殖是必不可少的。之前，我们和其他人报道了eIF5A在Lys47和/或Lys68位点乙酰化的生化证据，以及在Lys47位点乙酰化对其的负调控。我们还获得了多胺乙酰化酶亚精胺/精胺n1 -乙酰转移酶（SSAT1）在hypusine残基上乙酰化eIF5A的证据，以及SSAT1介导的hypusine乙酰化使eIF5A失活的证据。

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.