The Post-translational Synthesis of Hypusine In eIF5A

eIF5A 中 Hypusine 的翻译后合成

• 批准号: 8743739
• 负责人: MYUNG H PARK
• 金额: $ 108.63万
• 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/8743739
• 关键词: Acetylation; Adenoviruses; Amino Acids; Anabolism; Binding; Biochemical; Biochemical Reaction; Biological Assay; Cattle; Cell Proliferation; Cell physiology; Cells; DNA; DNA Synthesis Inhibition; DNA biosynthesis; Disease; Enzymes; Eukaryota; Eukaryotic Cell; Eukaryotic Initiation Factors; Genes; Genetic Transcription; Goals; Hela Cells; Hydroxylation; Image; Intervention; Ion Exchange; Lysine; Mammalian Cell; Messenger RNA; Metabolic; Methods; Modification; Molecular Mechanisms of Action; Pathway interactions; Phospholipids; Polyamines; Polyribosomes; Post-Translational Protein Processing; Property; Protein Biosynthesis; Protein Synthesis Inhibition; Proteins; Puromycin; Putrescine; RNA; Regulation; Reporting; Role; Side; Site; Specificity; Spermidine; Spermidine/Spermine N1-Acetyltransferase; Spermine; Testis; Translation Initiation; Translations; amino group; analog; cell growth; cellular imaging; cellular transduction; deoxyhypusine; deoxyhypusine monooxygenase; deoxyhypusine synthase; hypusine; inhibitor/antagonist; macromolecule; mutant; norspermine; novel; overexpression; polycation; vector

In previous studies we have identified eIF5A as the only cellular protein that contains an unusual amino acid, hypusine N-epsilon-(4-amino-2-hydroxybutyl)lysine, and established that hypusine biosynthesis occurs by two sequential enzymatic reactions: i) deoxyhypusine synthesis and ii) deoxyhypusine hydroxylation. We have cloned and characterized the structural and catalytic properties of the two enzymes of the hypusine pathway, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). We and others have demonstrated that hypusine modification is essential for the activity of eIF5A and for mammalian cell proliferation. Previously, we reported biochemical evidence for acetylation of eIF5A at Lys47 and its negative regulation by this acetylation. We have also obtained evidence for selective acetylation of hypusinated eIF5A, but not nonhypusinated eIF5A, by a polyamine metabolic enzyme, spermidine/spermine acetyltransferase 1 (SSAT1). The site of eIF5A acetylation by SSAT1 was identified as terminal amino group of the hypusine side chain by ion exchange chromatographic separation. The bovine testis eIF5A acetylated by SSAT1 was inactive in methionyl-puromycin synthesis assay indicating the importance of the basic side chain of hypusine residue in eIF5A activity. In the course of studying the molecular interaction between eIF5A and SSAT1, we observed suppression of expression of GFP-eIF5A , GFP or any other cotransfected gene by overexpression of SSAT1. SSAT1 activity was required for this inhibition, as overexpression of inactive SSAT1 mutant enzyme did not exert the same effect. The inhibition was not at transcription, since GFP mRNA or GFP-eIF5A mRNA level did not decrease upon cotransfection with SSAT1. This was confirmed by single cell imaging of mRNAs by Stellaris RNA FISH method. Furthermore, single cell fluorescent imaging of synthesis of DNA and RNA revealed that depletion of polyamines by SSAT1 overexpression does not cause inhibition of synthesis of DNA or RNA. Addition of polyamine analogs, such as Bis-ethyl-norspermine (BENSpm) which are not substrates for SSAT1, restored expression of GFP or GFP-eIF5A in the SSAT1 overexpressing cells, indicating that a lack of of polyamines is the underlying cause for inhibition of protein synthesis. In order to characterize biochemical changes in SSAT1 overexpressing cells, we used SSAT1 adenovirus to overexpress SSAT1 in all 293T or HeLa cells. Upon transduction with AdenoSSAT1, a rapid depletion of spermidine and spermine followed by an increase in putrescine and N1-acetylspermidine was observed. Within 24h of AdenoSSAT1 transduction, a total arrest in protein synthesis and cell growth occured, whereas there was no inhibition in cells transduced with AdenoGFP vector. Furthermore, comparison of polysome profiles showed a loss of polysomes in the AdenoSSAT1 transduced cells, but not in the AdenoGFP transduced cells, suggesting a mechanism of a block in translation initiation. Taken together, these findings suggest that the most important function of polyamines in eukaryotic cell growth is to promote translation.

在先前的研究中，我们已经鉴定eIF5A为含有不寻常的氨基酸羟腐胺赖氨酸N-β-（4-氨基-2-羟丁基）赖氨酸的唯一细胞蛋白，并确定羟腐胺赖氨酸生物合成通过两个连续的酶促反应发生：i）脱氧羟腐胺赖氨酸合成和ii）脱氧羟腐胺赖氨酸羟基化。我们已经克隆并表征了羟腐胺赖氨酸途径的两种酶，脱氧羟腐胺赖氨酸合成酶（DHS）和脱氧羟腐胺赖氨酸羟化酶（DOHH）的结构和催化性质。我们和其他人已经证明羟腐胺赖氨酸修饰对于eIF5A的活性和哺乳动物细胞增殖是必不可少的。 以前，我们报道了eIF5A在Lys47处乙酰化的生物化学证据及其通过这种乙酰化的负调控。我们还获得了证据羟腐胺赖氨酸化eIF5A，而不是nonhypusinated eIF5A，由多胺代谢酶，亚精胺/精胺乙酰转移酶1（SSAT1）的选择性乙酰化。通过离子交换色谱分离，将SSAT 1对eIF5A乙酰化的位点鉴定为羟腐胺赖氨酸侧链的末端氨基。经SSAT 1乙酰化的牛睾丸eIF5A在甲硫氨酰嘌呤霉素合成试验中无活性，表明羟腐胺赖氨酸残基的碱性侧链在eIF5A活性中的重要性。 在研究eIF 5A和SSAT 1之间的分子相互作用的过程中，我们观察到SSAT 1的过表达会抑制GFP-eIF 5A、GFP或任何其他共转染基因的表达。这种抑制需要SSAT1活性，因为过表达失活的SSAT1突变酶不会产生相同的效果。这种抑制作用不存在于转录中，因为GFP mRNA或GFP-eIF5A mRNA水平在与SSAT 1共转染后没有降低。这通过Stellaris RNA FISH方法对mRNA进行单细胞成像得到证实。此外，DNA和RNA合成的单细胞荧光成像显示，SSAT 1过表达消耗多胺不会导致DNA或RNA合成的抑制。添加多胺类似物，如不是SSAT 1底物的双乙基去甲精胺（BENSpm），恢复了SSAT 1过表达细胞中GFP或GFP-eIF5A的表达，表明缺乏多胺是抑制蛋白质合成的根本原因。为了表征SSAT 1过表达细胞中的生化变化，我们使用SSAT 1腺病毒在所有293 T或HeLa细胞中过表达SSAT 1。在用AdenoSSAT1转导后，观察到亚精胺和精胺的快速消耗，随后是腐胺和N1-乙酰亚精胺的增加。在AdenoSSAT1转导的24小时内，发生蛋白质合成和细胞生长的完全停滞，而在用AdenoGFP载体转导的细胞中没有抑制。此外，多核糖体谱的比较显示在AdenoSSAT1转导的细胞中多核糖体的损失，但在AdenoGFP转导的细胞中没有，这表明翻译起始中的阻断机制。总之，这些发现表明，多胺在真核细胞生长中最重要的功能是促进翻译。