The Post-translational Synthesis of Hypusine In eIF5A

在以前的研究中，我们已经确定eIF5A是唯一含有一种不寻常的氨基酸N-epsilon-(4-氨基-2-羟基丁基)赖氨酸的细胞蛋白质，并证实了亚硫氨酸的生物合成通过两个连续的酶反应进行：i)脱氧亚硫氨酸的合成和ii)脱氧亚硫氨酸的羟化。我们克隆并鉴定了亚精氨酸途径的两种酶--脱氧亚硫氨酸合成酶(DHS)和脱氧亚硫氨酸羟基酶(DOHH)的结构和催化性质。我们和其他人已经证明了亚精氨酸修饰对于eIF-5A的活性和哺乳动物细胞的增殖是必不可少的。以前，我们和其他人报道了eIF5A在Lys47和/或Lys68的乙酰化以及在Lys 47的乙酰化对其负调控的生化证据。我们还获得了多胺乙酰化酶亚精胺/精胺N1-乙酰转移酶(SSAT1)在亚精氨酸残基上乙酰化eIF5A的证据，以及SSAT1介导的亚精氨酸乙酰化使eIF5A失活的证据。 在研究eIF5A与SSAT1分子相互作用的过程中，我们观察到SSAT1对外源基因表达的强烈抑制作用。这种抑制依赖于SSAT1的活性，并且仅限于任何瞬时转基因基因的外源表达。这一过程似乎不涉及蛋白水解酶或核酸酶的激活，也与整个细胞多胺库的耗尽无关，这表明SSAT1具有新的功能和靶点。未观察到外源SSAT1对任何内源基因表达的影响，表明内源和外源基因表达途径的分子机制不同。 EIF5A是一种可能的翻译启动因子，可刺激甲硫酰嘌呤合成。然而，最近对酿酒酵母和黑腹葡萄球菌的遗传学研究表明，eIF5A在翻译的延伸步骤中发挥了作用。Hypusine修饰是eIF5A在翻译过程中的活性及其与核糖体结合所必需的。通过使用多胺生物合成的抑制剂二氟甲基鸟氨酸(DFMO)，我们获得的证据表明，多胺通过影响eIF2α和eIF4E-BP的磷酸化来调节转译启动。我们的数据来自被eIF5A siRNA和亚硫氨酸修饰抑制剂去除eIF5A的细胞，表明eIF5A在哺乳动物细胞翻译的延长步骤中发挥作用。 为了研究eIF5A亚型1和脱氧亚硫氨酸合成酶的生理功能，我们在小鼠身上进行了它们的基因打靶。我们使用了ES细胞系RRE174(Eif5a1+/-)和RRM039(DHPs+/-)，这两株细胞具有Eif5a1或DHPS基因的一个等位基因。从这些ES细胞中提取的基因组DNA进行聚合酶链式反应，证实每个基因都被破坏后，将RRE174和RRM039细胞注射到C57的囊胚中，并将注射的囊胚植入假孕C57雌性小鼠体内。每个克隆的雄性嵌合体与C57雌性交配产生刺鼠幼崽。基因靶向杂合子刺鼠(Eif5a1+/-，或DHPs+/-)似乎是正常的，没有表现出任何大体生长缺陷或表型。对杂合的雄鼠和雌鼠进行杂交，并对杂合杂交所生的幼鼠进行基因分型，以确定Eif5a1或DHPs的纯合子破坏是否致命。 在超过10个杂合杂交(Eif5a1+/-)的65只仔猪中，没有一只出生时携带Eif5a1纯合子KO基因。因此，Eif5a1基因的破坏必然会导致小鼠的胚胎死亡。在囊胚期(E3.5)发现Eif5a1-/-纯合子胚胎，但在后期未发现，表明Eif5a1-/-胚胎可存活至3.5天。 小鼠DHPS纯合子基因破坏的表型也是胚胎致死的。在11个杂合子Aggoti杂交后代的52只仔猪中，没有发现纯合子DHPS中断的幼崽。DHPS-/-纯合子胚胎在囊胚期(E3.5)才鉴定出来，但在后期没有鉴定到。这些结果表明eIF5A-1和DHS在小鼠早期胚胎发育中都起着重要的作用。

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.