HYPUSINE FORMATION ON EIF 5A--BIOCHEMISTRY AND FUNCTION

EIF 5A 上的前驱素形成--生物化学和功能

• 批准号: 2700421
• 负责人: KUANG Yu CHEN
• 金额: $ 24.73万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2700421
• 关键词: RNA binding protein; affinity chromatography; aminoacid analog; aminoacid biosynthesis; cell differentiation; cell growth regulation; developmental genetics; genetic library; genetic regulation; immunoprecipitation; intermolecular interaction; lysine; molecular cloning; neuroblastoma; polyamines; protein biosynthesis; protein purification; tissue /cell culture; transcription factor; translation factor; yeast two hybrid system

Hypusine formation on elF-5A precursor is by tar the most specific polyamine-dependent biological event in living cells. Deoxyhypusine synthase catalyzes the oxidative transfer of the aminobutyl moiety from spermidine to a unique lysine residue on elF-SA precursor to form deoxyhypusine residue which becomes hypusine after further hydroxylation. Deletion of either deoxyhypusine synthase or elF-5A gene in yeast gives a lethal phenotype. Although in vivo studies suggest that hypusine formation is tightly coupled to cell proliferation, the precise function of elF-5A and the physiological significance of hypusine formation are not clear. It has been suggested that elF-5A is the cellular target of HIV-1 viral protein Rev. We have obtained preliminary data indicating that modified eIF-5A, but not the unmodified precursor, caused gel mobility supershift of the Rev-RRE RNA complex, suggesting that deoxyhypusine/hypusine modification is required for the direct interaction between elF-5A and Rev. Rev is an RNA binding protein involved in the export of selected mRNAs. If the elF-5A interacting protein is Rev-like, it would imply that elF-5A may have a role in pre-mRNA processing and selective gene expression. We will use Rev and other Rev-like proteins (Rex, NS1 etc) as a model to gain insight of the specificity and structural requirement of these interactions, including the role of RNA species. We will use the yeast two-hybrid system, a powerful technique for studying protein- protein interaction, in conjunction with other more conventional methods, including co-immunoprecipitation, protein affinity chromatography photo-cross-inking and expression library probing, to search for the elF-5A interacting proteins. We have generated all necessary molecular tools (antibodies, cDNA clones, unmodified and modified recombinant elF-5As) for this purpose. The putative elF-5A interacting proteins identified by any of the above screening methods will be further characterized to confirm the interaction both in vitro and in vivo. Functional domains on elF- 5A, particularly the protein binding sites for Rev and for elF-5A interacting proteins will be defined by mutational analysis and site-specific cross-linking. Finally, the regulation and physiological function of elF-5A and its interacting proteins will be investigated in mouse neuroblastoma cells using pharmacological, histochemical and biochemical means. Preliminary studies have shown that specific inhibition of deoxyhypusine synthase could promote neuroblastoma differentiation. Possible effect of hypusine formation on the expression of cell cycle-dependent growth associated genes will be examined in normal human diploid fibroblasts. It is likely that elF-5A, mediated through its interacting proteins, may be involved in the regulation of a small class of genes essential for proliferation and for differentiation.

eIF-5A前体上的羟腐胺赖氨酸形成是通过焦油最特异性的 活细胞中依赖多胺的生物事件。脱氧羟 合成酶催化氨基丁基部分的氧化转移 从亚精胺到eIF-SA前体上独特的赖氨酸残基， 形成脱氧羟腐胺赖氨酸残基， 羟基化脱氧羟腐胺赖氨酸合酶或eIF-5A的缺失 酵母中的基因产生致死表型。尽管在体内研究中 表明羟腐胺赖氨酸形成与细胞紧密结合 细胞增殖、elF-5A的确切功能和细胞的生理功能， 羟腐胺赖氨酸形成意义尚不清楚。已经 表明eIF-5A是HIV-1病毒蛋白的细胞靶点 Rev.我们获得的初步数据表明， eIF-5A，而不是未修饰的前体，引起凝胶迁移 Rev-RRE RNA复合物的超位移，表明 需要脱氧羟腐胺赖氨酸/羟腐胺赖氨酸修饰以直接 eIF-5A和Rev. Rev之间的相互作用是RNA结合蛋白 参与选择性mRNA的输出。如果elF-5A 蛋白质是Rev样的，这意味着elF-5A可能在 前mRNA加工和选择性基因表达。我们将使用Rev 和其他Rev-样蛋白（雷克斯、NS 1等）作为模型获得 了解这些的特异性和结构要求， 相互作用，包括RNA种类的作用。我们将使用 酵母双杂交系统，一种研究蛋白质的强大技术， 蛋白质相互作用，结合其他更传统的 方法，包括免疫共沉淀，蛋白亲和 层析光交联和表达文库探测， 寻找eIF-5A相互作用蛋白。我们已经生成 所有必需的分子工具（抗体、cDNA克隆、未修饰的 和修饰的重组eIF-5A）。推定的 通过上述任一项鉴定的eIF-5A相互作用蛋白 筛选方法将进一步表征，以确认 在体外和体内的相互作用。eIF上的功能结构域- 5A的蛋白结合位点，特别是Rev和eIF-5A的蛋白结合位点 相互作用蛋白将通过突变分析来定义， 位点特异性交联。最后，规则和 eIF-5A及其相互作用蛋白的生理功能将 在小鼠神经母细胞瘤细胞中进行研究， 组织化学和生物化学方法。初步研究表明 特异性抑制脱氧羟腐胺赖氨酸合酶可以促进 神经母细胞瘤分化羟腐胺辛的可能影响 形成对细胞周期依赖性生长的表达 将在正常人类二倍体中检查相关基因 成纤维细胞elF-5A很可能通过其介导 相互作用的蛋白质，可能参与调节小的 一类对增殖和分化至关重要的基因。