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。脱氧亚硫氨酸合成酶和脱氧亚精氨酸羟基酶的抑制剂在哺乳动物细胞中具有很强的抗增殖作用。我们检测了脱氧硫辛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。这些化合物对脱氧次精氨酸羟基酶和脯氨酸羟基酶有抑制作用，并使细胞周期停滞于G1期。在Mimosine、2，2‘-联吡啶、去铁酮、去铁胺和环吡喃五种金属螯合抑制剂中，抗真菌药物环吡罗在两种模型中对这两种蛋白羟化酶、HUVEC增殖和血管生成的抑制作用最强。此外，这种化合物对一组人类癌细胞株具有很强的抗增殖作用。这些发现表明，环洛匹罗是治疗实体瘤的临床试验的有价值的候选药物。我们使用截短形式的eIF5A和突变的酶进行了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是Lys47、Lys50、His51、Pro82和Pro114。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。然而，这两个基因是差异调控的，在大多数人类细胞中，eIF5AI表达：只有在两个人类癌细胞株UACC1598和SW480中，eIF5AI和eIF5AII都被检测到。Northern和RT-PCR实验表明，EIF5A2基因的可译性较差是由于其3‘端非编码区。

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.