The Post-translational Synthesis of Hypusine In eIF5A

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。 以前，我们报道了eIF 5A在Lys 47处乙酰化的生物化学证据及其通过这种乙酰化的负调控。 我们还获得了多胺代谢酶，亚精胺/精胺乙酰转移酶1（SSAT 1）的eIF 5A的羟腐胺赖氨酸残基的选择性乙酰化的证据。 不含羟腐胺赖氨酸/脱氧羟腐胺赖氨酸残基的eIF 5A野生型或突变体蛋白均不作为SSAT 1的底物。经离子交换层析分离，鉴定eIF 5A的乙酰化氨基酸残基为乙酰基-羟腐胺赖氨酸或乙酰基-脱氧羟腐胺赖氨酸。在哺乳动物细胞中，与eIF 5A和SSAT 1载体共转染，eIF 5A和SSAT 1之间的大分子相互作用被证明通过共免疫沉淀，这表明乙酰化的特异性的分子基础。当将经SSAT 1乙酰化的牛睾丸eIF 5A的活性与未乙酰化的eIF 5A的活性进行比较时，乙酰化形式在甲硫氨酰-嘌呤霉素合成测定中无活性，表明羟腐胺赖氨酸残基的碱性侧链在eIF 5A活性中的重要性。SSAT 1可能通过羟腐胺赖氨酸残基的乙酰化来调节eIF 5A的活性。 羟腐胺赖氨酸修饰对于eIF 5A的活性和真核细胞增殖是必不可少的。然而，碱性羟腐胺赖氨酸残基在eIF 5A结构或其活性中的确切作用尚不清楚。研究eIF 5A（Hpu）、eIF 5A（Dhp）及其与修饰酶的复合物的结构以及确定eIF 5A在核糖体上的结合位点需要大量高纯度的蛋白质。然而，难以从哺乳动物组织中纯化足够量的含羟腐胺赖氨酸的eIF 5A。 我们利用多顺反子载体pST 39共表达eIF 5A，并利用修饰酶DHS和DOHH对eIF 5A（Lys）进行修饰。coli细胞。通过这种方法，我们已经生产了高纯度和高产量的eIF 5A（Hpu）以及中间体eIF 5A（Dhp）。在37 ℃诱导4 h快速产生的重组eIF 5A（Hpu）在甲硫氨酰嘌呤霉素合成试验中无活性，可能是由于蛋白质折叠不当。然而，在18 ℃过夜诱导的重组eIF 5A（Hpu）显示出与从牛睾丸纯化的天然eIF 5A相当的活性，而未羟腐胺赖氨酸化的eIF 5A前体eIF 5A（Lys）无活性。含脱氧羟腐胺赖氨酸的eIF 5A表现出部分活性。细菌表达的重组eIF 5A（Dhp）和eIF 5A（Hpu）将为eIF 5A的结构/功能研究提供有用的工具。 为了研究eIF 5A同种型和羟腐胺赖氨酸修饰酶的生理功能，我们使用ES细胞系RRE 174（Eif 5a-1 +/-）和RRM 039（Dhps +/-）在小鼠中进行它们的基因靶向，所述ES细胞系具有通过基因捕获方法破坏的每个基因的一个等位基因。基因靶向的杂合agglutinin小鼠（Eif 5a-1 +/-，或Dhps +/-）似乎是正常的，没有显示任何生长缺陷或表型。将杂合的无精子症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。没有出生的幼崽具有Eif 5a-1-/-或Dhps-/-的基因型，表明任一基因的纯合破坏是胚胎致死的。为了阐明胚胎致死的时间点，我们在发育阶段（E3.5，E6.5，E7.5和E8.5）培养胚胎，并通过PCR对其进行基因分型。 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-/-胚胎存活长达3.5天，但在6天后不能存活。Dhps-/-胚胎也是如此。这些发现表明eIF 5A-1和DHS在胚胎发育的早期阶段起着至关重要的作用。

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 eIF5A and for mammalian cell proliferation. Previously, we reported biochemical evidence for acetylation of eIF5A at Lys47 and its negative regulation by this acetylation. We have also obtained evidence for selective acetylation of hypusine residue of eIF5A by a polyamine metabolic enzyme, spermidine/spermine acetyltransferase 1 (SSAT1). None of the eIF5A wild type or mutant proteins not containing the hypusine/deoxyhypusine residue worked as a substrate for SSAT1. The amino acid residue of eIF5A acetylated by SSAT1 was identified as acetyl-hypusine or acetyl-deoxyhypusine by ion exchange chromatographic separation. In mammalian cells, cotransfected with eIF5A and SSAT1 vectors, macromolecular interaction between eIF5A and SSAT1 was demonstrated by co-immuno-precipitation, suggesting molecular basis for the specificity of this acetylation. When the activities of bovine testis eIF5A acetylated by SSAT1 was compared with that of non-acetylated eIF5A, the acetylated form was inactive in methionyl-puromycin synthesis assay indicating the importance of the basic side chain of hypusine residue in eIF5A activity. SSAT1 may regulate eIF5A activity by acetylation of its hypusine residue. Hypusine modification is essential for the activity of eIF5A and for eukaryotic cell proliferation. However, the exact role of the basic hypusine residue in eIF5A structure or in its activity is not known. A large quantity of highly pure proteins are needed for structural studies of eIF5A(Hpu), eIF5A(Dhp) and their complexes with modification enzymes and in determination of binding sites of eIF5A on ribosome. However, it is difficult to purify hypusine-containing eIF5A in sufficient quantity from mammalian tissues. We have used a polycistronic vector pST39 to coexpress eIF5A with the modification enzymes, DHS and DOHH for modification of eIF5A(Lys) in E. coli cells. By this method, we have produced eIF5A(Hpu) as well as the intermediate eIF5A(Dhp) in high purity and quantity. The recombinant eIF5A(Hpu) rapidly produced by induction at 37 C for 4 h was inactive in methionyl-puromycin synthesis assay, probably due to improper protein folding. However, recombinant eIF5A(Hpu) induced at 18 C overnight displayed comparable activity as the native eIF5A purified from bovine testis, whereas non-hypusinated eIF5A precursor, eIF5A(Lys) was inactive. Deoxyhypusine-containing eIF5A exhibited a partial activity. The bacterially produced recombinant eIF5A(Dhp) and eIF5A(Hpu) will be useful tools in structure/function studies of eIF5A. To investigate the physiological function of eIF5A isoforms and the hypusine modification enzymes, we performed their gene targeting in mice using the ES cell lines, RRE174 (Eif5a-1 +/-) and RRM039 (Dhps +/-) which have one allele of each gene disrupted by the gene trap method. The gene-targeted heterozygous agouti mice (Eif5a-1 +/-, or Dhps +/-) appeared to be normal and did not show any growth defects or phenotypes. The heterozygous agouti male and female mice were crossed and the pups born from the heterozygous intercrosses were genotyped. No pups were born with the genotype of Eif5a-1-/- or Dhps-/- indicating that homozygous disruption of either gene is embryonic lethal. To clarify the time point of embryonic lethality, we cultured the embryos at developmental stages (E3.5, E6.5, E7.5 and E8.5) and genotyped them by PCR. The Eif5a-1-/- homozygous embryo was identified on the blastocyst stage (E3.5), but not at later stage, indicating that Eif5a-1-/- embryo is viable up to 3.5 days, but not after 6 days. The same was true for Dhps-/- embryos. These findings demonstrate that eIF5A-1 and DHS play an essential role at the early stage of embryonic development.