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。我们还获得了证据羟腐胺赖氨酸化eIF 5A，而不是nonhypusinated eIF 5A，由多胺代谢酶，亚精胺/精胺乙酰转移酶1（SSAT 1）的选择性乙酰化。通过离子交换色谱分离，将SSAT 1对eIF 5A乙酰化的位点鉴定为羟腐胺赖氨酸侧链的末端氨基。经SSAT 1乙酰化的牛睾丸eIF 5A在甲硫氨酰嘌呤霉素合成试验中无活性，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。 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。 我们研究了原生动物寄生虫杜氏利什曼原虫（Leishmania donovani）DOHH功能的分子和结构基础，该原虫导致内脏利什曼病。洛杉矶DOHH基因全长981 bp，编码326个氨基酸。DOHH是一种HEAT重复蛋白，具有8个串联重复的螺旋对。四个保守的组氨酸-谷氨酸序列已被确定为金属配位位点。通过在大肠杆菌中异源表达DOHH，获得了一个42 kDa的带有His标签的重组蛋白。纯化的重组DOHH在体外有效地催化中间体eIF 5A-deoxyhypusine（eIF 5A-Dhp）的羟基化。L. donovani DOHH（LdDOHH）与其人同源物显示40.6%的序列同一性。L. donovani DOHH与人同源物的比较显示，在前者中存在两个显著的插入，对应于存在于连接蛋白质的N-和C-末端一半的可变环中的比对位置159-162（4个氨基酸残基）和174-183（10个氨基酸残基），后者存在于底物结合位点附近。缺失10个氨基酸长的插入将LdDOHH活性降低至野生型重组LdDOHH的14%。金属螯合剂环吡酮胺（CPX）和含羞草碱对L. donovani和DOHH活性。这些抑制剂对寄生虫酶比人类酶更有效。并对L. donovani DOHH和人同源物可用于基于结构设计抗寄生虫的选择性抑制剂。 EF-P是eIF 5A的细菌直向同源物，并且其不经历羟腐胺赖氨酸修饰。 最近，分别编码II类赖氨酰-tRNA合成酶和赖氨酸-2，3-氨基变位酶的截短同源物的两个细菌基因yjeA和yjeK已经涉及EF-P的修饰，以将特定的赖氨酸转化为假设的β-赖氨酰-赖氨酸。为了获得该假说的生化证据并评估这种修饰在EF-P活性中的作用，我们在大肠杆菌中过量表达了单独的EF-P或EF-P与YjeA和YjeK的组合。coli中表达。单独的EF-P、EF-P加YjeA和EF-P加YjeA加YjeK的过表达分别产生未修饰的EF-P、α-赖氨酰EF-P和β-赖氨酰EF-P。质谱分析证实了天然EF-P以及重组α-赖氨酰或β-赖氨酰EF-P蛋白中赖氨酸34处的赖氨酰修饰。 在天然EF-P和分离自共表达EF-P、YjeA和YjeK的大肠杆菌的重组EF-P的详尽链霉蛋白酶解物中鉴定了β-赖氨酰-赖氨酸异肽，但在衍生自单独编码EF-P或EF-P与YjeA一起编码的载体的蛋白质的酶解物中没有鉴定，这表明两种酶YjeA和YjeK，内源性EF-P和重组的β-赖氨酰-EF-P刺激的N-甲酰基-甲硫氨酰-嘌呤霉素合成是含有未修饰的EF-P和/或α-赖氨酰-EF-P的制剂的大约四倍。缺乏修饰位点赖氨酸的突变体（K34 A）是无活性的。这是EF-P在体内的β-裂解的生化证据和EF-P活性的这种修饰的要求的第一份报告。

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 hypusinated eIF5A, but not nonhypusinated eIF5A, by a polyamine metabolic enzyme, spermidine/spermine acetyltransferase 1 (SSAT1). The site of eIF5A acetylation by SSAT1 was identified as terminal amino group of the hypusine side chain by ion exchange chromatographic separation. The bovine testis eIF5A acetylated by SSAT1 was inactive in methionyl-puromycin synthesis assay indicating the importance of the basic side chain of hypusine residue in eIF5A activity. 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. We investigated the molecular and structural basis of the function of DOHH from the protozoan parasite, Leishmania donovani, which causes visceral leishmaniasis. The L. donovani DOHH gene is 981 bp and encodes a putative polypeptide of 326 amino acids. DOHH is a HEAT-repeat protein with eight tandem repeats of -helical pairs. Four conserved histidine-glutamate sequences have been identified that may act as metal coordination sites. A 42 kDa recombinant protein with a His-tag was obtained by heterologous expression of DOHH in Escherichia coli. Purified recombinant DOHH effectively catalyzed the hydroxylation of the intermediate, eIF5A-deoxyhypusine (eIF5A-Dhp), in vitro. L. donovani DOHH (LdDOHH) showed 40.6% sequence identity with its human homolog. The alignment of L. donovani DOHH with the human homolog shows that there are two significant insertions in the former, corresponding to the alignment positions 159-162 (four amino acid residues) and 174-183 (ten amino acid residues) which are present in the variable loop connecting the N- and C-terminal halves of the protein, the latter being present near the substrate binding site. Deletion of the ten-amino-acid-long insertion decreased LdDOHH activity to 14% of the wild type recombinant LdDOHH. Metal chelators like ciclopirox olamine (CPX) and mimosine significantly inhibited the growth of L. donovani and DOHH activity in vitro. These inhibitors were more effective against the parasite enzyme than the human enzyme. The structural differences between the L. donovani DOHH and the human homolog may be exploited for structure based design of selective inhibitors against the parasite. EF-P is a bacterial ortholog of eIF5A and it does not undergo hypusine modification. Recently, two bacterial genes, yjeA and yjeK, encoding truncated homologs of class II lysyl-tRNA synthetase and of lysine-2,3-aminomutase, respectively, have been implicated in the modification of EF-P to convert a specific lysine to a hypothetical beta-lysyl-lysine. To obtain biochemical evidence for the hypothesis and to assess the role of this modification in EF-P activity, we have over-expressed EF-P alone, or in combination with YjeA and YjeK in E. coli using a polycistronic vector. Over-expression of EF-P alone, EF-P plus YjeA, and EF-P plus YjeA plus YjeK resulted in unmodified EF-P, alpha-lysyl EF-P and beta-lysyl EF-P, respectively. Mass spectrometric analyses confirmed the lysyl modification at lysine 34 in native EF-P as well as in recombinant alpha-lysyl or beta-lysyl EF-P proteins. The beta-lysyl-lysine isopeptide was identified in the exhaustive pronase digests of native EF-P and recombinant EF-P isolated from E.coli coexpressing EF-P, YjeA and YjeK, but not in the digests of proteins derived from the vectors encoding EF-P alone or EF-P together with YjeA, indicating that both enzymes, YjeA and YjeK, are required for beta-lysylation of EF-P. Endogenous EF-P and the recombinant beta-lysyl-EF-P stimulated N-formyl-methionyl-puromycin synthesis approximately four-fold over the preparations containing unmodified EF-P and/or alpha-lysyl-EF-P. The mutant lacking the modification site lysine (K34A) was inactive. This is the first report of biochemical evidence for the beta-lysylation of EF-P in vivo and the requirement for this modification for the activity of EF-P.