The Post-translational Synthesis of Hypusine In eIF5A

在以前的研究中，我们已经确定eIF5A是唯一含有一种不寻常的氨基酸N-epsilon-(4-氨基-2-羟基丁基)赖氨酸的细胞蛋白质，并证实了亚硫氨酸的生物合成通过两个连续的酶反应进行：i)脱氧亚硫氨酸的合成和ii)脱氧亚硫氨酸的羟化。我们克隆并鉴定了亚精氨酸途径的两种酶--脱氧亚硫氨酸合成酶(DHS)和脱氧亚硫氨酸羟基酶(DOHH)的结构和催化性质。我们和其他人已经证明了亚硫氨酸修饰对于eIF5A的活性和哺乳动物细胞的增殖是必不可少的。此前，我们报道了Lys47上eIF5A乙酰化的生化证据以及这种乙酰化对eIF5A的负调控。我们还获得了多胺代谢酶亚精胺/精胺乙酰转移酶1(SSAT1)选择性乙酰化过低的eIF5A而不是非过低的eIF5A的证据。经离子交换层析分离，eIF5A被SSAT1乙酰化的位置为亚硫氨酸侧链的末端氨基。经SSAT1乙酰化的牛睾丸eIF5A在甲硫氨基-嘌呤霉素合成实验中无活性，表明亚硫氨酸残基的碱性侧链在eIF5A活性中起重要作用。 在研究eIF5A和SSAT1之间的分子相互作用过程中，我们观察到SSAT1的过表达抑制了GFP-eIF5A、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突变酶不会产生同样的效果。由于GFP和GFP-eIF5A在与SSAT1共转染后，GFP-eIF5A和GFP-eIF5A的mRNA水平没有下降，因此这种抑制不是在转录上。用Stellaris RNA FISH方法对mRNAs进行单细胞成像证实了这一点。此外，DNA和RNA合成的单细胞荧光成像显示，SSAT1过表达耗尽多胺并不会抑制DNA或RNA的合成。加入不是SSAT1底物的多胺类似物，如双乙基去甲精胺(BENSpm)，在SSAT1过表达的细胞中恢复了GFP或GFP-eIF5A的表达，表明多胺的缺乏是抑制蛋白质合成的根本原因。为了研究SSAT1过表达细胞的生化变化，我们利用SSAT1腺病毒在293T和HeLa细胞中过表达SSAT1。经AdenoSSAT1转导后，亚精胺和亚精胺迅速耗尽，腐胺和N1-乙酰亚精胺增加。在转导AdenoSSAT1后的24小时内，蛋白质合成和细胞生长完全停止，而转导AdenoGFP载体的细胞则没有受到抑制。此外，多聚体图谱的比较显示，在转导AdenoSSAT1的细胞中有多聚体丢失，但在AdenoGFP转导的细胞中没有，这表明翻译起始受阻的机制。综上所述，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。

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.