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: 2405844
• 负责人: KUANG Yu CHEN
• 金额: $ 24.16万
• 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/2405844
• 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.

在ELF-5A前体上形成的Hypusine是焦油形成的最特异的 活细胞中的多胺依赖生物事件。脱氧亚硫氨酸 合成酶催化氨基丁基部分的氧化转移 从亚精胺到ELF-SA前体上独特的赖氨酸残基 形成脱氧亚精氨酸残留物，进一步形成亚精氨酸 羟化作用。脱氧亚硫氨酸合成酶或ELF-5A的缺失 酵母中的基因给出了致命的表型。尽管体内研究 提示催产素的形成与细胞紧密相连 增殖、ELF-5A的精确功能与生理学 苏氨酸的形成意义尚不清楚。一直以来 提示ELF-5A是HIV-1病毒蛋白的细胞靶点 Rev.我们已经获得了初步数据，表明修改后的 EIF-5A，但不是未经修饰的前体，引起凝胶流动性 REV-RRE RNA复合体的超位移，表明 需要对脱氧亚硫氨酸/亚精氨酸进行直接修饰 ELF-5A与REV的相互作用是一种RNA结合蛋白 参与选定信使核糖核酸的出口。如果精灵-5A相互作用 蛋白质是REV样的，这意味着ELF-5A可能在 前信使核糖核酸的加工和选择性基因表达。我们将使用Rev 和其他REV样蛋白(REX、NS1等)作为模型获得 洞察这些项目的特殊性和结构要求 相互作用，包括RNA物种的作用。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，与其他更传统的 方法，包括免疫共沉淀，蛋白质亲和力 层析光交叉墨迹和表达文库探测， 寻找ELF-5A相互作用蛋白。我们已经产生了 所有必要的分子工具(抗体、cDNA克隆、未修饰 和经修饰的重组ELF-5A)。推定的 由上述任一种鉴定的ELF-5A相互作用蛋白 筛查方法将进一步表征，以确认 体外和体内的相互作用。精灵上的功能域- 5A，特别是REV和ELF-5A的蛋白结合位点 相互作用的蛋白质将通过突变分析和 站点特定的交叉链接。最后，监管和 ELF-5A及其相互作用蛋白的生理功能 在小鼠神经母细胞瘤细胞中进行药理学研究， 组织化学和生化方法。初步研究表明 脱氧亚硫氨酸合成酶的特异性抑制可以促进 神经母细胞瘤分化。催产素的可能作用 细胞周期依赖性生长表达的形成 相关基因将在正常人类二倍体中进行检测 成纤维细胞。很可能是精灵5A，通过它的 相互作用的蛋白质，可能参与了小分子的调节 对增殖和分化至关重要的一类基因。