Post Translational Synthesis Of Hypusine In Eif5a

Eif5a 中 Hypusine 的翻译后合成

• 批准号: 6535277
• 负责人: MYUNG HEE PARK
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6535277
• 关键词: aminoacid; aminoacid biosynthesis; cell growth regulation; cell proliferation; enzyme mechanism; enzyme structure; human tissue; ligase; polyamines; posttranslational modifications; recombinant proteins; spermidine; translation factor

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.

我们已经鉴定出 eIF5A 是唯一含有一种不寻常的氨基酸，hypusine [Nepsilon-(4-amino-2-羟丁基)lysine]的细胞蛋白，并且已经确定 hypusine 生物合成是通过两个连续的酶促反应在翻译后发生的。在第一步中，脱氧马尿苷合酶催化聚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，以形成中间体脱氧马尿苷残基。在后一步骤中，该中间体通过金属酶脱氧山茱萸碱羟化酶转化为山茱萸碱。 Hypusine 对于 eIF-5A 的活性和真核细胞增殖至关重要。因此，马尿苷生物合成步骤为干预真核细胞增殖提供了新的靶标。除了人脱氧马尿苷合成酶与 NAD 复合物的 X 射线晶体结构外，还确定了酶、NAD 和抑制剂 GC7 之间的三元复合物的新结构。这些结构揭示了 NAD 结合位点和推测亚精胺结合的活性位点袋。通过定点诱变评估了预测参与 NAD、亚精胺以及催化关键氨基酸结合的许多氨基酸的作用。亚精胺结合位点的分子建模应有助于开发脱氧马尿苷合成酶的特异性抑制剂，该抑制剂可用作抗增殖剂。我们测试了脱氧马尿碱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山茱萸羟化酶和脯氨酸羟化酶，并导致细胞周期停滞在 G1 期。在两种模型测定中，含羞草碱、2,2?-联吡啶、去铁酮、去铁胺和环吡酮五种金属螯合抑制剂中，抗真菌药物环吡酮对两种蛋白羟化酶、HUVEC增殖和血管生成的抑制最有效。此外，这种化合物对一组人类癌细胞系具有很强的抗增殖作用。这些发现表明环吡酮是治疗实体瘤临床试验的有价值的候选药物。