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

在以前的研究中，我们证明了多阳离子多胺在翻译调节的主要作用，通过过度表达的多胺分解代谢酶，亚精胺/精胺乙酰转移酶1（SAT 1）在HEK 293细胞。在通过用腺SAT 1病毒转导快速耗尽多胺后，蛋白质合成在起始水平被抑制，而RNA或DNA的合成没有或几乎没有抑制。在目前的时期，我们已经获得的证据，增强蛋白质合成的细胞中增加细胞腐胺和亚精胺，由于稳定的鸟氨酸脱羧酶（ODC）的活性，在多胺生物合成的第一步酶。 这一发现来自全基因组siRNA筛选，该筛选旨在鉴定沉默将增强重组蛋白生产的基因。沉默OAZ 1（编码ODC抗酶1的基因）持续增加重组蛋白的产量，包括可溶性、膜结合和分泌蛋白。OAZ 1的沉默在翻译水平而不是在转录水平上增加了重组蛋白的产生，这强调了多胺在翻译调控中的重要作用。 我们还研究了eIF 5A的功能，通过蛋白质组学分析的eIF 5A缺失的HeLa细胞的腺-eIF 5A的shRNA转导，以确定细胞的途径和蛋白质，直接或间接地影响eIF 5A。我们在iTRAQ实验1、2和3中分别鉴定了3810、1258和2750种独特蛋白质（2种独特肽，置信水平>95%），在所有三次运行中通常检测到972种蛋白质。其中，选择在Ad-eIF 5A-shRNA转导72和96小时具有显著改变的水平（蛋白质比率1.5或0.66，p值0.05）的104种蛋白质用于进一步分析聚脯氨酸的发生和功能本体分类。在eIF 5A缺失的细胞中，聚脯氨酸基序的含量与蛋白水平之间没有观察到一致的关系。对显著改变的蛋白质的功能本体的分析揭示了在eIF 5A耗尽的细胞中显著上调或下调的特定生物过程，并将蛋白质折叠鉴定为受eIF 5A耗尽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（尤其是p50-ATF 6）水平的增加得到证实。我们的数据从这些公正的，定量的，蛋白质组学分析表明，eIF 5A的耗尽导致内质网应激，未折叠的蛋白质反应和上调的伴侣蛋白在HeLa细胞中的表达。

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.