The Post-translational Synthesis of Hypusine In eIF5A

eIF5A 中 Hypusine 的翻译后合成

• 批准号: 6814502
• 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/6814502
• 关键词: aminoacid; aminoacid biosynthesis; cell growth regulation; cell proliferation; enzyme mechanism; enzyme structure; human tissue; ligase; northern blottings; polyamines; polymerase chain reaction; 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. Inhibitors of hypusine biosynthesis exert strong anti-proliferative effects in mammalian cells. Ciclopirox, an anti-fungal drug and inhibitor of deoxyhypusine hydroxylase exhibits anti-angiogenic activity and inhibits growth of various human cancer cell lines including the cervical cancer line SiHa, by causing a cell cycle arrest at G1/S boundary. We conducted structure/function studies of eIF5A, using truncated forms of eIF5A and mutant forms with single amino acid substitutions . 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, Gly49, Lys50, His51, Gly52, Lys55, Leu91, Leu101 and Glu143. 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. Although the two human eIF5A isoforms are highly similar in sequence (84% identity) they exhibit distinct immunoreactivity and kinetic values as substrates of deoxyhypusine synthase. Furthermore, the two eIF5A genes are differentially regulated. Whereas eIF5A-1 is highly expressed in all human cells, eIF5A-2 protein is not normally expressed. eIF5A-2 expression is limited to some specific tissues or cancer cells such as UACC1598 and SW480 and it has been suggested as an oncogene. Northern and RTPCR experiments suggest that the low expression of eIF5A-2 isoform is largely due to the poor translatability of eIF5A-2 mRNA and that its 3' UTR is responsible for the inefficient translation.

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的生物合成发生后，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。羟腐胺赖氨酸生物合成的抑制剂在哺乳动物细胞中发挥强的抗增殖作用。环吡酮是一种抗真菌药物和脱氧羟腐胺赖氨酸羟化酶抑制剂，通过在G1/S边界引起细胞周期停滞，表现出抗血管生成活性并抑制包括宫颈癌细胞系SiHa在内的各种人类癌细胞系的生长。我们使用截短形式的eIF 5A和具有单个氨基酸取代的突变形式进行了eIF 5A的结构/功能研究。结果表明，eIF 5A的氨基端和羧基端的10个氨基酸是其活性所必需的。迄今为止，鉴定为对eIF 5A功能至关重要的氨基酸残基是Lys 47、Gly 49、Lys 50、His 51、Gly 52、Lys 55、Leu 91、Leu 101和Glu 143。我们比较了两个人eIF 5A基因的表达和功能。酵母中的互补研究表明，这两个基因编码真正的eIF 5A蛋白具有相似的基本细胞功能。尽管两种人eIF 5A同种型在序列上高度相似（84%同一性），但它们作为脱氧羟腐胺赖氨酸合酶的底物表现出不同的免疫反应性和动力学值。此外，两个eIF 5A基因的差异调节。尽管eIF 5A-1在所有人类细胞中高度表达，但eIF 5A-2蛋白通常不表达。eIF 5A-2的表达局限于某些特定的组织或癌细胞，如UACC 1598和SW 480，并被认为是一种癌基因。北方和RTPCR实验表明eIF 5A-2亚型的低表达主要是由于eIF 5A-2 mRNA的可翻译性差，其3' UTR是导致eIF 5A-2翻译效率低下的原因。