ESSENTIAL CELLULAR FUNCTION OF HYPUSINE IN ELF 5A

真核蛋白质翻译起始因子5A(eIF-5A)含有一个 并且似乎是唯一含有这种物质的细胞蛋白质 独特的氨基酸。Hypusine是在翻译后通过转移产生的 将多胺亚精胺的丁胺部分转化为赖氨酰残基 在eIF-5A前体中形成脱氧亚硫氨酸，然后羟化 形成扁桃体。 含有亚硫氨酸的蛋白eIF-5A的确切生理作用 目前还不得而知。然而，众所周知，催产素是至关重要的。 用于真核细胞的增殖。严格保护自然资源 围绕亚硫氨酸残基的12个氨基酸的序列进一步 强调了这种残留物的重要性。我们调查了 与底物相关的苏氨酸合成的专一性 以eIF-5A前体蛋白片段为底物的蛋白质 脱氧亚硫氨酸合成酶，亚精氨酸生物合成的第一种酶。这些 是由特定的内蛋白酶或重组人产生的 删除亚克隆。结果确定了EIF-5A的最小结构域 脱氧亚硫氨酸合成所需前体蛋白Phe 30-Asp 80 并深入了解eIF-5A之间的分子相互作用 前体和脱氧亚精氨酸合酶。 为了鉴定eIF-5A与之相互作用的细胞蛋白 发挥其生物活性，我们开创了一种亲和方法 他们的孤立。多组氨酸标记的人eIF-5A前体蛋白 Tag-EC-eIF-5A)。这个 通过对His-Tag-EC-TAG的修饰，制备了含有脱氧亚精氨酸的形式。 EIF-5A在脱氧亚精氨酸合酶反应中的表达。组氨酸标记的蛋白质 结合到Ni(II)-NTA-琼脂糖柱被用作亲和基质 分离和鉴定与之特异相互作用的细胞蛋白 EIF-5A。 在另一个实验室最近的一项研究中，据报道eIF-5A是一种 HIV复制中REV功能所需的宿主细胞因子- 1.我们目前正在研究亚硫氨酸修饰在脑出血中的作用。 EIF-5A与REV的相互作用及HIV-1的可能干预 通过抑制亚精氨酸的合成进行复制。

Eukaryotic protein translation initiation factor 5A (eIF-5A) contains one residue of hypusine and appears to be the only cellular protein with this unique amino acid. Hypusine is produced post-translationally by transfer of the butylamine portion of the polyamine spermidine to a lysyl residue in the eIF-5A precursor to form deoxyhypusine followed by hydroxylation to form hypusine. The precise physiological role of the hypusine-containing protein eIF-5A is yet unknown. However, it is well established that hypusine is vital for eukaryotic cell proliferation. The strict conservation of the sequence of twelve amino acids surrounding the hypusine residue further emphasizes the importance of this residue. We investigated the basis for the specificity of hypusine synthesis with respect to the substrate protein using fragments of eIF-5A precursor protein as substrates for deoxyhypusine synthase, the first enzyme in hypusine biosynthesis. These were generated either by specific endoproteinases or by recombinant deletion subcloning. The results define the minimum domain of the eIF-5A precursor protein required for deoxyhypusine synthesis as Phe 30-Asp 80 and provide insight into the molecular interaction between eIF-5A precursor and deoxyhypusine synthase. In an effort to identify cellular proteins with which eIF-5A interacts to exert its biological activity, we have originated an affinity method for their isolation. Polyhistidine-tagged human eIF-5A precursor protein (His- tag-ec-eIF-5A) was produced by utilizing pET-24b vector. The deoxyhypusine-containing form was prepared by modification of His-tag-ec- eIF-5A in the deoxyhypusine synthase reaction. The his-tagged proteins bound to a Ni (II)-NTA-agarose column are being used as an affinity matrix to isolate and identify cellular proteins that specifically interact with eIF-5A. In a recent study from another laboratory, eIF-5A was reported to be a host cellular factor required for Rev function in the replication of HIV- 1. We are currently investigating the role of hypusine modification in the interaction of eIF-5A and Rev and the possible intervention of HIV-1 replication through inhibition of hypusine synthesis.