The Post-translational Synthesis of Hypusine In eIF5A

在之前的研究中，我们已经通过多胺分解代谢酶亚精胺/精胺乙酰转移酶1 （SSAT1）在hypusine残基上选择性乙酰化eIF5A，获得了eIF5A失活的证据。通过在哺乳动物细胞中共表达eIF5A和SSAT1之间的分子相互作用，我们研究了SSAT1过表达导致亚精胺和精胺完全消耗的细胞多胺的功能。在这种情况下，我们观察到GFP- eif5a， GFP或任何其他共转染基因的表达完全抑制。抑制作用发生在翻译水平而非转录水平，因为GFP mRNA或GFP- eif5a mRNA在与SSAT1共转染后没有减少。利用腺病毒在293T细胞中过表达SSAT1导致亚精胺和精胺的快速消耗，蛋白质合成受阻，细胞生长抑制和凋亡细胞死亡。添加多胺类似物，如α -甲基亚精胺和二甲基精胺，它们不是SSAT1的底物，可以恢复翻译和细胞生长，这为多胺的消耗导致生长停滞和细胞死亡提供了证据。MDL72527抑制多胺氧化酶并不能恢复细胞生长，这表明生长停滞不是由多胺分解代谢加速引起的氧化应激引起的。膜联蛋白V染色和碘化丙啶荧光活化细胞分选仪（FACS）分析显示，AdSAT1转导后48小时凋亡明显增加。核断裂和caspase 3活性的增加、聚ADP核糖聚合酶（PARP）的裂解以及细胞色素c从线粒体释放到细胞质进一步表明这些多胺缺失细胞通过线粒体内在途径凋亡。此外，adsat1转导细胞的电镜图像显示了与凋亡相关的形态学变化，包括核断裂、细胞质收缩、线粒体改变、空泡化和膜起泡。与AdSAT1转导相比，对照GFP腺病毒（AdGFP）转导的影响较小，这表明细胞凋亡主要是由SAT1的过表达引起的，而不是腺病毒转导本身。这些数据提供了强有力的证据，证明多胺亚精胺和精胺对细胞活力至关重要。

In previous studies, we have obtained evidence for inactivation of eIF5A by selective acetylation of eIF5A at hypusine residue by a polyamine catabolic enzyme, spermidine/spermine acetyltransferase 1 (SSAT1). Investigation of the molecular interaction between eIF5A and SSAT1 by coexpression in mammalian cells led us to study the functions of cellular polyamines as SSAT1 overexpression leads to total depletion of spermidine and spermine. Under this circumstance, we observed total inhibition of expression of GFP-eIF5A, GFP or any other cotransfected gene. The inhibition was at the level of translation but not at transcription, since GFP mRNA or GFP-eIF5A mRNA did not decrease upon cotransfection with SSAT1. Overexpression of SSAT1 in 293T cells by use of adenovirus resulted in a rapid depletion of spermidine and spermine, arrest in protein synthesis, inhibition of cell growth and apoptotic cell death. Addition of polyamine analogs, such as alpha-methylspermidine and dimethylspermine that are not substrates for SSAT1, restored translation and cell growth, providing evidence that depletion of polyamines caused growth arrest and cell death. Inhibition of polyamine oxidases by MDL72527 did not restore cell growth suggesting that growth arrest was not induced by oxidative stress resulting from accelerated polyamine catabolism. Annexin V staining and propidium iodide Fluorescent Activated Cell Sorter (FACS) analyses showed a clear increase in apoptosis at 48 h after AdSAT1 transduction. Nuclear fragmentation and an increase in caspase 3 activity, Poly ADP ribose polymerase (PARP) cleavage and release of cytochrome c from mitochondria into cytoplasm further suggest apoptosis by the intrinsic mitochondrial pathway in these polyamine-depleted cells. Moreover, electron microscopy images of AdSAT1-transduced cells revealed morphological changes commonly associated with apoptosis, including nuclear fragmentation, cytoplasm shrinkage, mitochondrial alteration, vacuolization and membrane blebbing. The effects of control GFP adenovirus (AdGFP) transduction were minor compared to those of AdSAT1 transduction, suggesting that the apoptosis was mainly caused by an over-expression of SAT1 rather than adenoviral transduction per se. These data provide strong evidence that the polyamines spermidine and spermine are vital for cell viability.