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硫氨酸修饰对于eIF5A的活性和哺乳动物细胞的增殖是必不可少的。此前，我们报道了Lys47上eIF5A乙酰化的生化证据以及这种乙酰化对eIF5A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(SSAT1)选择性乙酰化eIF5A的亚精氨酸残基的证据。 不含亚精氨酸/脱氧亚硫氨酸残基的eIF5A野生型或突变型蛋白均不能作为SSAT1的底物。经SSAT1乙酰化的eIF5A的氨基酸残基经离子交换层析分离鉴定为乙酰亚硫氨酸或乙酰脱氧亚硫氨酸。在与eIF5A和SSAT1共转染的哺乳动物细胞中，免疫共沉淀证实了eIF5A和SSAT1之间的大分子相互作用，这可能是这种乙酰化的特异性的分子基础。将SSAT1乙酰化的牛睾丸eIF5A的活性与未乙酰化的eIF5A进行比较，发现乙酰化的eIF5A在甲硫基-嘌呤霉素合成实验中没有活性，表明亚硫氨酸残基的碱性侧链在eIF5A活性中的重要性。SSAT1可能通过其亚硫氨酸残基的乙酰化来调节eIF5A的活性。 Hypusine修饰对于eIF5A的活性和真核细胞的增殖是必不可少的。然而，碱性亚硫氨酸残基在eIF5A结构或其活性中的确切作用尚不清楚。为了研究eIF5A(HPU)、eIF5A(DHP)及其与修饰酶的络合物的结构和确定eIF5A与核糖体的结合部位，需要大量高纯度的蛋白质。然而，从哺乳动物组织中大量纯化含有亚硫氨酸的eIF5A是很困难的。我们利用多顺反子载体pST39与修饰酶DHS和DOHH在大肠杆菌中共表达eIF5A(Lys)。用这种方法，我们得到了高纯度、高质量的eIF5A(HPU)和中间体eIF5A(DHP)。重组eIF5A(HPU)在37℃诱导4h后，在甲硫氨酰-嘌呤霉素合成实验中失活，可能是由于蛋白质折叠不当所致。然而，在18℃下诱导的重组eIF5A(HPU)显示出与从牛睾丸中纯化的天然eIF5A相似的活性，而非低糖eIF5A前体eIF5A(Lys)则没有活性。含有脱氧亚硫氨酸的eIF5A具有部分活性。细菌产生的重组eIF5A(DHP)和eIF5A(HPU)将成为研究eIF5A结构和功能的有用工具。 为了研究eIF5A亚型和亚硫氨酸修饰酶的生理功能，我们用ES细胞株RRE174(Eif5a-1+/-)和RRM039(DHPs+/-)进行了它们的基因打靶实验，这两个细胞株的每个基因都有一个等位基因。基因靶向杂合子刺鼠(Eif5a-1+/-或DHPS+/-)似乎是正常的，没有表现出任何生长缺陷或表型。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。没有幼崽出生时携带Eif5a-1-/-或DHps-/-基因，这表明这两个基因的纯合子破坏都是胚胎致命的。为了弄清胚胎致死的时间点，我们对发育阶段(E3.5、E6.5、E7.5和E8.5)的胚胎进行了培养，并用聚合酶链式反应对它们进行了基因分型。Eif5a-1-/-纯合子胚胎在囊胚期(E3.5)被鉴定出来，但在后期没有发现，这表明Eif5a-1-/-胚胎在3.5天后仍能存活，但在6天后不能存活。DHPS-/-胚胎也是如此。这些发现表明eIF5A-1和DHS在胚胎发育的早期阶段起着重要的作用。

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 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.