The Post-translational Synthesis Of Hypusine In Eif5a

我们已经确定eIF5A是唯一含有不寻常氨基酸的细胞蛋白，hypusine [neplsilon -（4-氨基-2-羟基丁基）赖氨酸]，并且已经确定hypusine的生物合成是在翻译后通过两个连续的酶促反应进行的。在第一步中，脱氧hypusine合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上，形成中间产物脱氧hypusine残基。在后一步中，这种中间体通过金属酶脱氧hypusine羟化酶转化为hypusine。Hypusine是eIF-5A活性和真核细胞增殖所必需的。脱氧hypusine合成酶和脱氧hypusine羟化酶抑制剂在哺乳动物细胞中具有较强的抗增殖作用。我们测试了脱氧hypusine羟化酶抑制剂对人静脉内皮细胞（HUVEC）增殖和血管生成的影响。这些化合物抑制脱氧hypusine羟化酶和脯氨酸羟化酶，导致G1期细胞周期阻滞。在5种金属螯合抑制剂（氨莫辛、2,2'-二吡啶基、去铁嘧啶、去铁胺和环匹罗）中，抗真菌药物环匹罗对两种蛋白羟化酶、HUVEC增殖和血管生成的抑制作用在两个模型实验中最为有效。此外，这种化合物对一组人类癌细胞系有很强的抗增殖作用。这些发现表明环匹罗是治疗实体瘤临床试验中有价值的候选药物。我们使用eIF5A的截短形式和突变酶进行了eIF5A的结构/功能研究。结果表明，eIF5A的活性离不开氨基端或羧基端的10个氨基酸。目前发现的对eIF5A功能至关重要的氨基酸残基有Lys47、Lys50、His51、Pro82和Pro114。我们比较了两种人类eIF5A基因的表达和功能。酵母中的互补研究表明，这两个基因编码的eIF5A蛋白具有相似的基本细胞功能。然而，这两个基因受到不同的调控，在大多数人类细胞中，eIF5AI只表达一种异构体：只有在UACC1598和SW480两种人类癌细胞中，eIF5AI和eIF5AII都被检测到。Northern和RTPCR实验表明，EIF5A2 mRNA的可译性较差是由于其3' UTR。

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. The inhibitors of deoxyhypusine synthase and deoxyhypusine hydroxylase exert strong antiproliferative effects in mammalian cells. We have tested the effects of inhibitors of deoxyhypusine hydroxylase on human vein endothelial cell (HUVEC) proliferation and angiogenesis. These compounds inhibited deoxyhypusine hydroxylase and proline hydroxylase, and caused cell cycle arrest in G1. Of the five metal chelating inhibitors i.e. mimosine, 2,2'-dipyridyl, deferiprone, deferoxamine and ciclopirox, the antifungal drug ciclopirox was the most effective in the inhibition of the two protein hydroxylases, HUVEC proliferation and angiogenesis in two model assays. Furthermore, this compound exerts strong antiproliferative effects on a panel of human cancer cell lines. These findings suggest that ciclopirox is a valuable candidate for clinical trials in the treatment of solid tumors. We conducted structure/function studies of eIF5A, using truncated forms of eIF5A and mutant enzymes. 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, Lys50, His51, Pro82 and Pro114. 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. However, the two genes are differentially regulated and in most human cells, one isoform, eIF5AI is expressed: Only in two human cancer lines, UACC1598 and SW480, both eIF5AI and eIF5AII are detected. Northern and RTPCR experiments suggest that the poor translatability of EIF5A2 mRNA is due to its 3' UTR.