The Post-translational Synthesis of Hypusine In eIF5A and role of polyamines in cell growth and death

eIF5A 中 Hypusine 的翻译后合成以及多胺在细胞生长和死亡中的作用

• 批准号: 9339226
• 负责人: MYUNG H PARK
• 金额: $ 101.17万
• 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/9339226
• 关键词: Acetyltransferase; Affect; Amino Acid Motifs; Anabolism; Binding; Biological Process; Cell Death; Cell Proliferation; Cell physiology; Cells; Classification; DNA; DNA biosynthesis; Data; Disease; Enzymes; Eukaryota; Eukaryotic Cell; Eukaryotic Initiation Factors; Genes; Goals; Hela Cells; Intervention; Lysine; Membrane; Modification; Molecular Chaperones; Molecular Mechanisms of Action; Ontology; Ornithine Decarboxylase; Pathway interactions; Peptides; Phospholipids; Polyamines; Post-Translational Protein Processing; Production; Protein Biosynthesis; Proteins; Proteomics; Putrescine; RNA; RNA Synthesis Inhibition; RNA chemical synthesis; Recombinant Proteins; Role; Running; Signaling Molecule; Small Interfering RNA; Specificity; Spermidine; Spermidine/Spermine N1-Acetyltransferase; Spermine; TNFRSF5 gene; Translational Regulation; Translations; Up-Regulation; Viral Proteins; cell growth; deoxyhypusine monooxygenase; deoxyhypusine synthase; design; endoplasmic reticulum stress; genome-wide; hypusine; inhibitor/antagonist; macromolecule; novel; ornithine decarboxylase antizyme; overexpression; polycation; polyproline; protein folding; research study; response; small hairpin RNA

In previous studies, we demonstrated the primary role of the polycationic polyamines in translational regulation by overexpression of a polyamine catabolic enzyme, spermidine/spermine acetyltransferase 1 (SAT1) in HEK293 cells. Upon rapid depletion of polyamines by transduction with the adeno SAT1 virus, protein synthesis was inhibited at the level of initiation, while no or little inhibition of synthesis of RNA or DNA occurred. In the current period, we have obtained evidence for enhanced protein synthesis in cells with increased cellular putrescine and spermidine, due to stabilization of ornithine decarboxylase (ODC) activity, the first step enzyme in polyamine biosynthesis. This finding resulted from a genome-wide siRNA screen that was designed to identify genes whose silencing would enhance recombinant protein production. Silencing of OAZ1, the gene that encodes the ODC antizyme 1, consistently increased production of recombinant proteins, including soluble, membrane-bound and secreted proteins. Silencing of OAZ1 increased the recombinant protein production at the translational level, not at the transcriptional level, underscoring the important role of polyamines in translational regulation. We also investigated the function of eIF5A by proteomic analyses of HeLa cells depleted of eIF5A by adeno-eIF5A shRNA transduction, to identify cellular pathways and proteins that are directly or indirectly influenced by eIF5A. We identified 3810, 1258 and 2750 unique proteins (with 2 unique peptides with >95% confidence level) in iTRAQ experiment 1, 2 and 3, respectively, and 972 proteins commonly detected in all three runs. Of these, 104 proteins with significantly altered levels (protein ratio 1.5 or 0.66, p value 0.05) at 72, and 96h of Ad-eIF5A-shRNA transduction were selected for further analyses for polyproline occurrence and functional ontology classification. No consistent relationship was observed between the content of polyproline motifs and the protein levels in eIF5A-depleted cells. Analyses of the functional ontology of the significantly altered proteins revealed specific biological processes that are prominently up-or down-regulated in eIF5A-depleted cells and identified protein folding as the major cellular process affected by the depletion of eIF5A. Activation of the unfolded protein response (UPR) was confirmed by increases in levels of UPR signaling molecules, especially p50-ATF6. Our data from these unbiased, quantitative, proteomic analyses demonstrate that the depletion of eIF5A leads to endoplasmic reticulum stress, an unfolded protein response and up-regulation of chaperone expression in HeLa cells.

在以前的研究中，我们通过在HEK293细胞中过表达多胺分解代谢酶亚精胺/精胺乙酰转移酶1(SAT1)，证明了多阳离子多胺在翻译调控中的主要作用。在转导多胺后，蛋白质合成在起始水平受到抑制，而RNA和DNA的合成没有或几乎没有受到抑制。目前，由于鸟氨酸脱羧酶(ODC)活性的稳定，我们已经获得了细胞腐胺和亚精胺增加的细胞蛋白质合成增强的证据，鸟氨酸脱羧酶是多胺生物合成的第一步酶。这一发现是全基因组siRNA筛查的结果，该筛查旨在识别沉默会促进重组蛋白生产的基因。沉默OAZ1，编码ODC抗酶1的基因，持续增加重组蛋白的产生，包括可溶性、膜结合和分泌蛋白。沉默OAZ1增加了翻译水平上的重组蛋白产量，而不是转录水平上的重组蛋白产量，这突显了多胺在翻译调控中的重要作用。我们还通过对通过Adeno-eIF5A shRNA转导而缺失eIF5A的HeLa细胞的蛋白质组学分析，研究了eIF5A的功能，以确定eIF5A直接或间接影响的细胞通路和蛋白质。在iTRAQ实验1、2和3中，我们分别鉴定了3810、1258和2750个独特的蛋白质(其中2个独特的肽具有95%的置信度)，以及在所有三个运行中普遍检测到的972个蛋白质。在这些蛋白质中，选择了在72小时和96小时有显著变化(蛋白质比1.5或0.66，p值0.05)的104个蛋白质，用于进一步分析多聚脯氨酸的存在和功能本体论分类。在eIF5A缺失的细胞中，多聚脯氨酸基序的含量与蛋白质水平之间没有一致的关系。对显著改变的蛋白质的功能本体论的分析揭示了在eIF5A缺失的细胞中显著上调或下调的特定生物学过程，并发现蛋白质折叠是eIF5A缺失影响的主要细胞过程。未折叠蛋白反应(UPR)的激活被UPR信号分子水平的增加所证实，尤其是p50-ATF6。我们从这些无偏见的、定量的蛋白质组分析中获得的数据表明，eIF5A的缺失导致了内质网压力、未折叠的蛋白质反应和HeLa细胞中伴侣蛋白的表达上调。