The Post-translational Synthesis of Hypusine In eIF5A

eIF5A 中 Hypusine 的翻译后合成

• 批准号: 8148627
• 负责人: MYUNG H PARK
• 金额: $ 84.39万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8148627
• 关键词: hypusine

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.

在先前的研究中，我们已经鉴定eIF 5A为含有不寻常的氨基酸羟腐胺赖氨酸N-β-（4-氨基-2-羟丁基）赖氨酸的唯一细胞蛋白，并确定羟腐胺赖氨酸生物合成通过两个连续的酶促反应发生：i）脱氧羟腐胺赖氨酸合成和ii）脱氧羟腐胺赖氨酸羟基化。我们已经克隆并表征了羟腐胺赖氨酸途径的两种酶，脱氧羟腐胺赖氨酸合成酶（DHS）和脱氧羟腐胺赖氨酸羟化酶（DOHH）的结构和催化性质。我们和其他人已经证明，羟腐胺赖氨酸修饰是必不可少的活性的eIF-5A和哺乳动物细胞增殖。 先前，我们和其他人报道了eIF 5A在Lys 47和/或Lys 68处乙酰化及其通过Lys 47处乙酰化的负调节的生化证据。我们还获得了多胺乙酰化酶，亚精胺/精胺N1-乙酰转移酶（SSAT 1）乙酰化eIF 5A在羟腐胺赖氨酸残基的证据，并通过SSAT 1介导的羟腐胺赖氨酸乙酰化eIF 5A的失活。 在研究eIF 5A和SSAT 1之间的分子相互作用的过程中，我们观察到SSAT 1对外源基因表达的强烈抑制。这种抑制依赖于SSAT 1活性，并且仅限于任何瞬时转染基因的外源表达。这一过程似乎不涉及蛋白酶或核酸酶的激活，也与细胞内多胺库的耗竭无关，提示SSAT 1的新功能和靶点。外源性SSAT 1对任何内源性基因表达均无影响，表明内源性与外源性基因表达途径的分子机制存在差异。 eIF 5A是刺激甲硫氨酰嘌呤霉素合成的推定翻译起始因子。然而，最近的遗传研究在S。cerevisiae和D.黑腹果蝇的研究表明eIF 5A在翻译的延伸步骤中起作用。eIF 5A的翻译活性及其与核糖体的结合需要羟腐胺赖氨酸修饰。通过使用多胺生物合成的抑制剂二氟甲基鸟氨酸（DFMO），我们已经获得证据表明多胺通过其对eIF 2 α和eIF 4 E-BP的磷酸化的影响来调节transaltion起始。我们从eIF 5A SiRNA和羟腐胺赖氨酸修饰抑制剂耗尽eIF 5A的细胞中获得的数据表明，eIF 5A在哺乳动物细胞翻译的延伸步骤中发挥作用。 为了研究eIF 5A亚型1和脱氧羟腐胺赖氨酸合成酶的生理功能，我们在小鼠中进行了基因打靶。 我们使用ES细胞系RRE 174（Eif 5a 1 +/-）和RRM 039（Dhps +/-），其具有通过基因捕获方法破坏的Eif 5a 1或Dhps基因的一个等位基因。通过从这些ES细胞分离的基因组DNA的PCR确认各基因的破坏后，将RRE 174和RRM 039细胞注射到C57的胚泡中，并将注射的胚泡植入假怀孕的C57雌性小鼠中。将每个克隆的雄性嵌合体与C57雌性交配以产生无角幼体幼仔。基因靶向的杂合agglutinin小鼠（Eif 5a 1 +/-或Dhps+/-）似乎是正常的，没有显示任何总体生长缺陷或表型。将杂合的agglutinase雄性和雌性小鼠杂交，并对杂合杂交产生的幼崽进行基因分型，以确定Eif 5a 1或Dhps的纯合破坏是否致命。 在10次以上杂合杂交（Eif 5a 1 +/-）所生的65只幼仔中，没有出生具有Eif 5a 1纯合KO基因型的幼仔。 因此，Eif 5a 1基因破坏必然导致小鼠胚胎死亡。 Eif 5a 1-/-纯合胚胎在胚泡期（E3.5）被鉴定，但在后期未被鉴定，表明Eif 5a 1-/-胚胎可存活长达3.5天。 Dhps纯合基因破坏小鼠的表型也是胚胎致死的。在11个杂合杂交组合的52只幼仔中，未发现Dhps纯合突变，在囊胚期（E3.5）发现了Dhps -/-纯合胚胎，但在囊胚后期未发现。这些结果表明，eIF 5A-1和DHS在小鼠早期胚胎发育中起重要作用。