The Post-translational Synthesis of Hypusine In eIF5A

eIF5A 中 Hypusine 的翻译后合成

• 批准号: 7146117
• 负责人: MYUNG HEE PARK
• 金额: --
• 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/7146117
• 关键词: aminoacid analog; aminoacid biosynthesis; biomarker; cell line; cell proliferation; cytogenetics; enzyme activity; enzyme inhibitors; gene expression profiling; human tissue; hydroxylation; liquid chromatography mass spectrometry; neoplastic cell culture for noncancer research; oxidoreductase; point mutation; polyamines; posttranslational modifications; protein isoforms; proteomics; translation factor; two dimensional gel electrophoresis

We previously 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 post-translational enzymatic reactions: i) deoxyhypusine synthesis and ii) deoxyhypusine hydroxylation. We cloned cDNA for deoxyhypusine synthase and characterized its structural and catalytic properties and demonstrated that hypusine modification is essential for the activity of eIF-5A and for eukaryotic cell proliferation. In this period (2004-2005), we have reported evidence for the independent roles of eIF5A and polyamines in cell proliferation and have identified a candidate gene, DRG-1 that mediates the effects of deoxyhypusine hydroxylase inhibitor, mimosine, on cell cycle arrest. Independent roles of eIF5A and polyamines in cell proliferation Polyamines are essential for cell proliferation. Since polyamines largely exist as polyamine-RNA complexes, their proliferative effects are presumed to be due to stimulation of protein synthesis. Polyamine spermidine is also required for activation of eIF5A through hypusine modification. In cells treated with inhibitors of polyamine biosynthesis, it was not clear whether growth inhibition was due to deprivation of polyamines, active eIF5A or both. The respective roles of active, hypusine-containing eIF5A and polyamines in cell proliferation were examined in mouse mammary carcinoma FM3A cells treated with an inhibitor of deoxyhypusine synthase, N1-guanyl-1,7-diaminoheptane (GC7), or with an inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine (DFMO), or with DFMO plus an inhibitor of spermine synthase, N1-(3-aminopropyl)cyclohexylamine (APCHA). Growth inhibition was observed after treatment with GC7 or with DFMO, due to reduction of either eIF5A or polyamines. The inhibition of growth observed in cells treated with both DFMO and APCHA, before a notable reduction in eIF5A, is probably due to a decrease in the total polyamines. The results indicate that a decrease in either active eIF5A or the total polyamine level leads to inhibition of cell growth, and that eIF5A and polyamines are independently involved in the regulation of cell growth. Structure/Function Studies of eIF5A-1 eIF5A is highly conserved throughout the eukaryotic kingdom. In order to understand the structural requirements of eIF5A for its cellular function, we generated variant forms of human eIF5A-1, with truncations of 6, 11 and 21 amino acids from either the NH2- or COOH-terminus, or with single amino substitutions of conserved amino acid residues, and tested the truncated or mutated human eIF5A-1 genes for their ability to support the growth of yeast. The results indicate that human eIF5A-1 with 6 amino acids deleted from the NH2- or COOH terminus are functional, but those with truncations of eleven or more residues from either end showed substantial or total loss of activity. The amino acid residues thus far identified to be critical for eIF5A functions are Lys47, Gly49, Lys50, Gly52, His51, Lys55A and E143. Differential expression of eIF5A-1 and eIF5A-2 in human cancer cells Of the two human eIF5A genes reported, normally only one isoform (eIF5A-1) is expressed in most human cell lines. eIF5A-1 and eIF5A-2 genes are differentially expressed in mammalian cells. Whereas eIF5A-1 mRNA and protein are constitutively expressed in all human cells, eIF5A-2 mRNAs (0.6-5.5 kb) are expressed in a tissue-specific manner. eIF5A-2 protein was detectable only in the colorectal and ovarian cancer-derived cell lines SW-480 and UACC-1598, which showed high over-expression of eIF5A-2 mRNAs. The multiple forms of eIF5A-2 mRNA, with varying lengths of the 3'-UTR were identified as products of one gene resulting from the use of different polyadenylation signals (AATAAA) in the 3'-UTR. These multiple eIF5A-2 mRNAs appeared to show similar sttability. The eIF5A-1 and -2 precursors were modified comparably in UACC-1598 cells and both isoform proteins were similarly stable. Comparison of expression levels of mRNA and protein of the two isoforms suggests that translation efficiency of eIF5A-2 mRNAs is much lower than that of the eIF5A-1 mRNA, probably due to 5'- and 3'-UTR of eIF5A-2 mRNAs. Although either human eIF5A gene can complement growth of a yeast strain in which the yeast eIF5A genes were disrupted (suggesting functional similarity of the two isoforms in supporting growth of yeast), the two eIF5A isoforms seem to have evolved specialized functions in higher eukaryotes. The differences in gene-expression patterns, physical characteristics and distinct amino acid sequences in the C-terminal domain imply differentiated, tissue-specific functions of the eIF5A-2 isoform in the mammalian organism and in cancer. The effects of deoxyhypusine hydroxylase inhibitor, mimosine on the expression of DRG-1 L-mimosine, an inhibitor of deoxyhypusine hydroxylase, causes cell cycle arrest at late G1 and has been reported to affect translation of mRNAs of the cyclin-dependent kinase inhibitor p27, eIF3a p170, and ribonucleotide reductase M2. To determine additional genes involved in the mimosine effects, proteomic analysis was performed on mimosine-treated cells, by two-dimensional gel electrophoresis followed by identification of the altered protein spots by LC-tandem mass spectrometry. Three altered protein spots were identified as differentiation-related gene 1 (DRG-1), deoxyhypusine-containing eIF5A intermediate and mature eIF5A (hypusine form). The reduction of the mature eIF5A with a concomittant increase in the deoxyhypusine-containing intermediate is due to inhibition of cellular deoxyhypusine hydroxylase by mimosine. Mimosine treatment caused an early induction of expression of DRG-1. The mimosine-induced expression of DRG-1 was mainly attributable to increased transcription, likely by the c-Jun/AP-1 transcription factor, and may be an important contributing factor in cell cycle arrest in G1.

我们之前发现eIF5A是唯一含有一种不寻常的氨基酸，hypusine [N-epsilon-（4-氨基-2-羟基丁基）赖氨酸]的细胞蛋白，并确定了hypusine的生物合成发生在两个连续的翻译后酶反应：i)脱氧hypusine合成和ii)脱氧hypusine羟基化。我们克隆了脱氧hypusine合成酶的cDNA，并对其结构和催化特性进行了表征，证明了hypusine修饰对于eIF-5A的活性和真核细胞的增殖是必不可少的。在此期间（2004-2005），我们报道了eIF5A和多胺在细胞增殖中的独立作用的证据，并确定了一个候选基因DRG-1，该基因介导脱氧hypusine羟化酶抑制剂（mimosine）对细胞周期阻滞的影响。