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

在之前的研究中，我们通过在HEK293细胞中过表达一种多胺分解代谢酶，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节中的主要作用。腺SAT1病毒转导多胺迅速耗尽后，蛋白质合成在起始水平受到抑制，而RNA或DNA合成没有或很少受到抑制。目前，我们已经获得证据表明，由于鸟氨酸脱羧酶（ODC）活性的稳定，多胺生物合成的第一步酶——腐胺和亚精胺增加了细胞中蛋白质合成的增强。这一发现来自全基因组siRNA筛选，该筛选旨在鉴定沉默可提高重组蛋白生产的基因。OAZ1（编码ODC抗酶1的基因）的沉默持续增加重组蛋白的产生，包括可溶性蛋白、膜结合蛋白和分泌蛋白。OAZ1的沉默增加了重组蛋白在翻译水平的产生，而不是在转录水平，强调了多胺在翻译调控中的重要作用。我们还通过对eIF5A通过腺-eIF5A shRNA转导缺失eIF5A的HeLa细胞进行蛋白质组学分析，研究了eIF5A的功能，以确定直接或间接受eIF5A影响的细胞通路和蛋白。我们在iTRAQ实验1、2和3中分别鉴定出3810、1258和2750种独特蛋白（其中2种独特肽，95%置信水平为>），在所有三次运行中分别检测到972种常见蛋白。其中，104个蛋白在Ad-eIF5A-shRNA转导72和96小时时水平显著改变（蛋白比值为1.5或0.66，p值0.05），用于进一步分析脯氨酸的发生和功能本体分类。在eif5a缺失的细胞中，聚脯氨酸基序的含量与蛋白水平之间没有一致的关系。对这些显著改变的蛋白的功能本体的分析揭示了在eIF5A缺失的细胞中显著上调或下调的特定生物学过程，并确定了蛋白折叠是受eIF5A缺失影响的主要细胞过程。未折叠蛋白反应（UPR）的激活通过UPR信号分子，特别是p50-ATF6水平的增加得到证实。我们从这些无偏的、定量的蛋白质组学分析中获得的数据表明，eIF5A的缺失导致HeLa细胞内质网应激、未折叠的蛋白质反应和伴侣蛋白表达上调。

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.