Post Translational Synthesis Of Hypusine In Eif5a

我们已经确定eIF 5A作为唯一的细胞蛋白，含有一种不寻常的氨基酸，羟腐胺赖氨酸[Nepaline-（4-氨基-2-羟丁基）赖氨酸]，并已建立羟腐胺赖氨酸生物合成发生后，通过两个连续的酶促反应。在第一步中，脱氧羟腐胺赖氨酸合酶催化多胺亚精胺的丁胺部分转移至eIF-5A前体蛋白中的特定赖氨酸残基，以形成中间体脱氧羟腐胺赖氨酸残基。在后一步骤中，该中间体通过金属酶脱氧羟腐胺赖氨酸羟化酶转化为羟腐胺赖氨酸。羟腐胺赖氨酸对于eIF-5A的活性和真核细胞增殖是必需的。因此，羟腐胺赖氨酸生物合成步骤提出了干预真核细胞增殖的新靶点。除了与NAD复合的人脱氧羟腐胺赖氨酸合酶的X射线晶体结构之外，还确定了酶NAD和抑制剂GC 7之间的三元复合物的新结构。这些结构揭示了NAD结合位点和亚精胺被假定结合的活性位点口袋。通过定点诱变评估了预测参与NAD、亚精胺结合的一些氨基酸的作用，以及那些对催化至关重要的氨基酸的作用。亚精胺结合位点的分子模拟应该有助于开发可能用作抗增殖剂的脱氧羟腐胺赖氨酸合酶的特异性抑制剂。我们已经测试了脱氧羟腐胺赖氨酸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羟腐胺赖氨酸羟化酶和脯氨酸羟化酶，并导致细胞周期停滞在G1期。在含羞草碱这5种金属螯合抑制剂中，2，2？联吡啶、去铁酮、去铁胺和环吡酮，抗真菌药物环吡酮在两种模型测定中对两种蛋白羟化酶、HUVEC增殖和血管生成的抑制最有效。此外，该化合物对一组人类癌细胞系发挥强抗增殖作用。这些发现表明，环吡酮是一个有价值的候选人，在治疗实体瘤的临床试验。

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.