The Post-translational Synthesis of Hypusine In eIF5A

我们已经鉴定出eIF5A是唯一含有一种不寻常的氨基酸--苏氨酸[海王星-(4-氨基-2-羟基丁基)赖氨酸]的细胞蛋白，并证实了苏氨酸的生物合成是通过两个连续的酶反应在翻译后发生的。在第一步中，脱氧亚精氨酸合成酶催化多胺亚精胺的丁胺部分转移到eIF-5A前体蛋白中的特定赖氨酸残基，以形成中间体脱氧亚精氨酸残基。在后一步中，该中间体被金属酶脱氧亚精氨酸羟基酶转化为亚硫氨酸。Hypusine对于eIF-5A的活性和真核细胞的增殖是必不可少的。亚精氨酸生物合成的抑制剂在哺乳动物细胞中具有很强的抗增殖作用。环洛匹罗是一种抗真菌药物，也是脱氧亚硫氨酸羟化酶的抑制剂，它具有抗血管生成活性，并通过使细胞周期停滞在G1/S边界而抑制包括宫颈癌SiHA在内的多种人类癌细胞株的生长。我们使用eIF5A的截短形式和带有单一氨基酸取代的突变形式进行了eIF5A的结构/功能研究。结果表明，eIF5A活性离不开氨基末端和羧基末端的10个氨基酸。到目前为止，对eIF5A功能至关重要的氨基酸残基有Lys47、Gly49、Lys50、His51、Gly52、Lys55、Leu91、Leu101和Glu143。我们比较了这两个人eIF5A基因的表达和功能。在酵母中的互补研究表明，这两个基因编码真正的eIF5A蛋白，具有相似的基本细胞功能。尽管这两个人eIF5A亚型在序列上高度相似(84%的同源性)，但它们作为脱氧亚硫氨酸合成酶的底物表现出不同的免疫反应性和动力学价值。此外，这两个eIF5A基因是差异调控的。虽然eIF5A-1在所有人类细胞中都高度表达，但eIF5A-2蛋白不正常表达。EIF5A-2仅在某些特定组织或癌细胞中表达，如UACC1598和SW480，已被认为是一种癌基因。Northern和RT-PCR实验表明，eIF5A-2亚型的低表达在很大程度上是由于eIF5A-2mRNA的可译性差，其3‘非编码区是翻译效率低下的原因。

We have identified eIF5A as the only cellular protein that contains an unusual amino acid, hypusine [Nepsilon- (4-amino-2-hydroxybutyl)lysine], and have established that hypusine biosynthesis occurs posttranslationally by two sequential enzymatic reactions. In the first step deoxyhypusine synthase catalyzes the transfer of the butylamine moiety of the polyamine spermidine to a specific lysine residue in the eIF-5A precursor protein to form an intermediate, deoxyhypusine residue. In the latter step, this intermediate is converted to hypusine by a metalloenzyme deoxyhypusine hydroxylase. Hypusine is essential for the activity of eIF-5A and for eukaryotic cell proliferation. Inhibitors of hypusine biosynthesis exert strong anti-proliferative effects in mammalian cells. Ciclopirox, an anti-fungal drug and inhibitor of deoxyhypusine hydroxylase exhibits anti-angiogenic activity and inhibits growth of various human cancer cell lines including the cervical cancer line SiHa, by causing a cell cycle arrest at G1/S boundary. We conducted structure/function studies of eIF5A, using truncated forms of eIF5A and mutant forms with single amino acid substitutions . The results indicate that 10 amino acids either from the amino terminal or the carboxyl terminal are indispensible for eIF5A activity. The amino acid residues thus far identified to be critical for eIF5A function are Lys47, Gly49, Lys50, His51, Gly52, Lys55, Leu91, Leu101 and Glu143. We compared the expression and function of the two human eIF5A genes. Complementation studies in yeast indicate that both genes encode bona fide eIF5A proteins with similar basic cellular function. Although the two human eIF5A isoforms are highly similar in sequence (84% identity) they exhibit distinct immunoreactivity and kinetic values as substrates of deoxyhypusine synthase. Furthermore, the two eIF5A genes are differentially regulated. Whereas eIF5A-1 is highly expressed in all human cells, eIF5A-2 protein is not normally expressed. eIF5A-2 expression is limited to some specific tissues or cancer cells such as UACC1598 and SW480 and it has been suggested as an oncogene. Northern and RTPCR experiments suggest that the low expression of eIF5A-2 isoform is largely due to the poor translatability of eIF5A-2 mRNA and that its 3' UTR is responsible for the inefficient translation.