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。因此，hypusine生物合成步骤为干预真核细胞增殖提供了新的靶点。除了与NAD配合物中的人脱氧hypusine合酶的x射线晶体结构外，还确定了该酶与NAD和抑制剂GC7之间的三元配合物的新结构。这些结构揭示了NAD结合位点和一个活性位点袋，亚精胺可能在其中结合。一些氨基酸的作用被预测参与NAD的结合，亚精胺，和那些对催化至关重要的，通过位点定向诱变评估。亚精胺结合位点的分子模型应该有助于开发脱氧hypusine合成酶的特异性抑制剂，这些抑制剂可能作为抗增殖剂有用。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。在五种金属螯合抑制剂中，例如，2,2？在两种模型实验中，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均以环匹罗最有效。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。

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. Thus hypusine biosynthetic steps present novel targets for intervention in eukaryotic cell proliferation. In addition to the X-ray crystal structure of human deoxyhypusine synthase in a complex with NAD, a new structure for a ternary complex between the enzyme, NAD and the inhibitor, GC7, has been determined. These structures reveal NAD binding sites and an active site pocket where spermidine is presumed to bind. The role of a number of amino acids predicted to be involved in the binding of NAD, of spermidine, and those critical for the catalysis, was assessed by site-directed mutagenesis. Molecular modeling of the spermidine binding site should aid development of specific inhibitors of deoxyhypusine synthase that may be useful as anti-proliferative agents. 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.