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The Post-translational Synthesis of Hypusine In eIF5A

eIF5A 中 Hypusine 的翻译后合成

基本信息

批准号：
7733913
负责人：
MYUNG HEE PARK
金额：
$ 80.83万
依托单位：
NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
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/7733913
关键词：
Acetylation Amino Acids Anabolism Binding Biochemical Reaction Biological C-terminal Cell Proliferation Cell physiology Cells Charge Collaborations Complement Data Defect Disease Elements Engineering Enzymes Eukaryotic Cell Eukaryotic Initiation Factors Exhibits Failure Galactose Genes Glucose Goals Growth Hand Helix (Snails)Homologous Gene Human Hydroxylation Impairment In Vitro Intervention Isomerism Laboratories Leishmania mexicana Location Lysine Mammalian Cell Modeling Modification Molecular Conformation Molecular Mechanisms of Action Mutation N-terminal Nature Nucleic Acids Phenotype Polyamines Post-Translational Protein Processing Protein Biosynthesis Protein Synthesis Inhibition Protein Synthesis Inhibitors Proteins Puromycin Rate Reaction Regulation Reporting Ribosomes Role Saccharomyces cerevisiae Side Site Site-Directed Mutagenesis Specificity Spermidine Stereoisomer Structure Temperature Testing Translating Translations Yeasts analog base cell growth cell type comparative deoxyhypusine deoxyhypusine monooxygenase deoxyhypusine synthase eIF-5A ear helix hypusine inhibitor/antagonist interest mutant novel promoter protein folding translation factor yeast protein

项目摘要

In the previous studies, we have identified eIF5A as the only cellular protein that contains an unusual amino acid, hypusine Ne-(4-amino-2-hydroxybutyl)lysine, a modified lysine with 4 aminobutyl moiety derived from the polyamine, spermidine. We have established that hypusine biosynthesis occurs post-translationally by two sequential enzymatic reactions: i) deoxyhypusine synthesis and ii) deoxyhypusine hydroxylation. We demonstrated that hypusine modification is essential for the activity of eIF-5A and for mammalian cell proliferation. Thus, hypusine synthesis defines one specific function of polyamines in cell proliferation. In addition to hypusine synthesis, polyamines bind to nucleic acids and regulate cellular activities at the transcriptional, translational and posttranslational levels. In mammalian cells, this polycationic function by bulk polyamines as well as hypusine synthesis is required for cell proliferation. In this reporting period, (in collaboration with Dr. Herbert Tabors group) we have investigated the role of hypusine and polyamines in the yeast S. cerevisiae. We have also conducted the structure/function studies of eIF5A, and identified the key structural elements of eIF5A critical for its biological activity in supporting growth and protein synthesis. Furthermore, we have obtained strong evidence that eIF5A is indeed a factor essential for eukaryotic protein synthesis. Hypusine modification is the most important function of the polyamine spermidine in the yeast S. cerevisiae: We have investigated the role of eIF5A in yeast polyamine auxotroph strains. Spermidine and its derivative, hypusinated eIF5A, have been shown to be essential for the growth of S. cerevisiae. In wild type cells, 1 % of cellular spermidine is normally utilized for hypusine synthesis. S. cerevisiae polyamine auxotrophs defective in polyamine synthesis grow at a nearly normal rate at low concentrations of spermidine (10-8 M) in the medium. In spe2 cells grown in this medium, cellular spermidine is less than 0.1 % of the normal level (<2 M vs. 2 mM), but as much as 37 % of the spermidine is mobilized for hypusine synthesis. The portion of spermidine used for hypusine synthesis increases dramatically as cellular spermidine becomes limiting. The vital importance of the hypusine modification is further supported by a positive correlation between the ability of spermidine analogs to modify eIF5A to a functional protein and their ability to support growth of the spe2 null mutant. 1-methyl spermidine supported growth whereas caldine, aminopropylcadaverine, 8-methylspermidine did not. Of the R- and S- stereoisomers of 1-methyl spermidine, the S-isomer was consistently better than the R-isomer in supporting growth and as a substrate for deoxyhypusine synthase (unpublished results). The failure of caldine to support growth is likely due to its inability to modify eIF5A. On the other hand, aminopropylcadaverine and 8-methylspermidine, both of which can serve as substrates of DHS to modify eIF5A, failed to support growth, suggesting a stringent structural requirement for the hypusine/deoxyhypusine side chain in eIF5A activity. Taken together, these findings indicate that the hypusine modification of eIF5A is the most critical (and perhaps primary) function of spermidine in supporting the growth of S. cerevisiae. Mutational analyses of human and yeast eIF5A: Identification of amino acid residues critical for the biological activity of eIF5A and the hypusine modification: To investigate the features of eIF5A required for its activity, we generated 49 mutations in human eIF5A-1. Each highly conserved residue was targeted by single site mutation and truncations were made from N- or C-terminus. All mutant proteins were tested for complementing the growth of an S. cerevisiae eIF5A null strain. Only a few mutant eIF5As with substitutions at or near the hypusine modification site (K47D, G49A, K50A, K50D, K50I, K50R, G52A and K55A) or with truncation of 21 amino acids from either the N- or C-terminus failed to support growth. For the Lys50 substitution proteins and G52A and K55A, the inactivity is due to the lack or impairment of deoxyhypusine modification. In contrast, K47D and G49A were effective substrates for deoxyhypusine synthase, yet failed to support growth, suggesting critical roles of Lys47 and Gly49 in eIF5A biological activity, possibly in its interaction with downstream effector(s). Comparison of the three different mutants, K47A, K47D and K47R, offers an interesting clue to the role of Lys47. K47R supports growth as well as the wild type protein, and K47A does so at a reduced rate. In contrast, no growth is observed upon expression of the K47D mutant, indicating the importance of the basic charge at residue 47. In view of evidence for acetylation at this site and loss of activities in K47A and K47D, Lys47 acetylation may serve as a natural mechanism in the regulation of eIF5A activity. A comparative structural and functional characterization was also performed on S. cerevisiae eIF5A (in collaboration with Dr. Sandro Valentinis group). The tertiary structure of yeast eIF5A was modeled based on the structures of its Leishmania mexicana homologues and this model was used to predict the location of site-directed and randomly-generated mutations in the tertiary structure. Most of the 40 new mutants exhibit phenotypes that result from eIF5A protein-folding defects. Our data provide evidence that the C-terminal -helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension is not. Similar to the human eIF5A mutants, yeast eIF5A mutant strains containing substitutions surrounding the hypusine modification site (K51 in the yeast protein) were nonviable (G50A, G50D, H52A, H52D, G53A, G53D, H54D and K56D) or showed temperature-sensitive (K56A) phenotype. These findings demonstrate the stringent requirement for the hypusine loop sequence in the interaction of eIF5A not only with its modification enzymes but also with its downstream effectors. eIF5A is an essential translation factor: Despite the essential nature of eIF5A in eukaryotic cell proliferation, the precise cellular function of eIF5A has remained obscure for decades. eIF5A enhances methionyl-puromycin synthesis in a deoxyhypusine- and hypusine-dependent manner in vitro. eIF5A binds to actively translating ribosomes and conditional mutants of eIF5A are hypersensitive to protein synthesis inhibitors. We have investigated the role of eIF5A in translation using the yeast strain, UBHY-R (derived in the laboratory of Dr. John WB Hershey). In this strain, the two yeast eIF5A genes are inactivated and its growth depends on genetically engineered unstable fusion-eIF5A (UBR5A), expressed under the GAL promoter. Upon shift of this strain from a galactose medium (YPGal) to a glucose medium (YPD), UBR5A was rapidly degraded, protein synthesis declined and cell growth was inhibited. Expression of human wild type eIF5A in the UBHY-R strain restored protein synthesis and growth, whereas expression of inactive mutant eIF5A proteins was without effect. Further evidence for a direct role of eIF5A in translation was obtained from yeast temperature-sensitive eIF5A mutants. Two of the temperature-sensitive yeast strains produced stable mutant proteins, eIF5AK56A and eIF5AQ22H,L93F. Upon shift of these two strains to a restrictive temperature, rapid inhibition of protein synthesis was observed, presumably as a result of an altered conformation of the mutant proteins upon the temperature shift.

在之前的研究中，我们已经确定 eIF5A 是唯一含有一种不寻常氨基酸的细胞蛋白，即高尿苷 Ne-(4-氨基-2-羟丁基)赖氨酸，这是一种源自多胺亚精胺的具有 4 个氨基丁基部分的修饰赖氨酸。我们已经确定，高尿苷生物合成是通过两个连续的酶促反应在翻译后发生的：i) 脱氧高尿苷合成和 ii) 脱氧高尿苷羟基化。我们证明，hypusine 修饰对于 eIF-5A 的活性和哺乳动物细胞增殖至关重要。因此，马尿苷合成定义了多胺在细胞增殖中的一种特定功能。除了马尿苷合成外，多胺还与核酸结合并在转录、翻译和翻译后水平上调节细胞活性。在哺乳动物细胞中，细胞增殖需要大量多胺以及马尿苷合成的这种聚阳离子功能。在本报告期内，（与 Herbert Tabors 博士小组合作）我们研究了马尿苷和多胺在酿酒酵母中的作用。我们还进行了 eIF5A 的结构/功能研究，并确定了 eIF5A 对其支持生长和蛋白质合成的生物活性至关重要的关键结构元件。此外，我们获得了强有力的证据表明eIF5A确实是真核蛋白质合成所必需的因子。 Hypusine 修饰是酿酒酵母中多胺亚精胺最重要的功能：我们研究了 eIF5A 在酵母多胺营养缺陷型菌株中的作用。亚精胺及其衍生物，hypuslated eIF5A，已被证明对于酿酒酵母的生长至关重要。在野生型细胞中，1%的细胞亚精胺通常用于合成马尿嘧啶。多胺合成缺陷的酿酒酵母多胺营养缺陷型在培养基中亚精胺浓度低（10-8 M）时以几乎正常的速率生长。在这种培养基中生长的 spe2 细胞中，细胞亚精胺低于正常水平的 0.1%（<2 M 对 2 mM），但多达 37% 的亚精胺被动员用于亚马丁合成。随着细胞亚精胺变得有限，用于合成亚丁胺的亚精胺部分急剧增加。亚精胺类似物将 eIF5A 修饰为功能蛋白的能力与它们支持 spe2 无效突变体生长的能力之间的正相关性进一步支持了 hypusine 修饰的至关重要性。 1-甲基亚精胺支持生长，而caldine、氨丙基尸胺、8-甲基亚精胺则不支持生长。在 1-甲基亚精胺的 R- 和 S- 立体异构体中，S-异构体在支持生长和作为脱氧马尿苷合成酶的底物方面始终优于 R-异构体（未发表的结果）。 caldine 未能支持生长可能是由于其无法修饰 eIF5A。另一方面，氨丙基尸胺和8-甲基亚精胺（两者都可以作为DHS的底物来修饰eIF5A）未能支持生长，表明eIF5A活性中对马尿苷/脱氧马尿苷侧链有严格的结构要求。总而言之，这些发现表明 eIF5A 的后尿嘧啶修饰是亚精胺在支持酿酒酵母生长中最关键（也可能是主要）的功能。人类和酵母 eIF5A 的突变分析：鉴定对 eIF5A 生物活性至关重要的氨基酸残基和后尿嘧啶修饰：为了研究其活性所需的 eIF5A 特征，我们在人类 eIF5A-1 中产生了 49 个突变。每个高度保守的残基都通过单位点突变进行靶向，并从 N 或 C 末端进行截短。所有突变蛋白均经过测试以补充酿酒酵母 eIF5A 无效菌株的生长。只有少数在后尿嘧啶修饰位点（K47D、G49A、K50A、K50D、K50I、K50R、G52A 和 K55A）处或附近进行取代或从 N 端或 C 端截断 21 个氨基酸的突变型 eIF5A 无法支持生长。对于 Lys50 取代蛋白以及 G52A 和 K55A，失活是由于脱氧马尿苷修饰的缺乏或受损所致。相比之下，K47D 和 G49A 是脱氧马尿苷合酶的有效底物，但未能支持生长，表明 Lys47 和 Gly49 在 eIF5A 生物活性中发挥关键作用，可能与下游效应子相互作用。三种不同突变体 K47A、K47D 和 K47R 的比较为 Lys47 的作用提供了有趣的线索。 K47R 与野生型蛋白一样支持生长，而 K47A 的生长速度较低。相反，在 K47D 突变体表达时没有观察到生长，这表明残基 47 处的碱性电荷的重要性。鉴于该位点乙酰化以及 K47A 和 K47D 活性丧失的证据，Lys47 乙酰化可能作为调节 eIF5A 活性的天然机制。还对酿酒酵母 eIF5A 进行了比较结构和功能表征（与 Sandro Valentinis 博士小组合作）。酵母 eIF5A 的三级结构是根据其墨西哥利什曼原虫同源物的结构建模的，该模型用于预测三级结构中定点突变和随机生成的突变的位置。 40 个新突变体中的大多数表现出由 eIF5A 蛋白折叠缺陷引起的表型。我们的数据提供证据表明，酵母 eIF5A 中存在的 C 端螺旋是必需的结构元件，而 eIF5A N 端 10 个氨基酸延伸则不是。与人类 eIF5A 突变体类似，在后尿嘧啶修饰位点（酵母蛋白中的 K51）周围含有取代的酵母 eIF5A 突变株无法存活（G50A、G50D、H52A、H52D、G53A、G53D、H54D 和 K56D）或表现出温度敏感（K56A）表型。这些发现表明，eIF5A 不仅与其修饰酶相互作用，而且与其下游效应子相互作用，对前叶嘧啶环序列有严格的要求。 eIF5A 是一种重要的翻译因子：尽管 eIF5A 在真核细胞增殖中具有重要性质，但几十年来 eIF5A 的精确细胞功能仍然不清楚。 eIF5A 在体外以脱氧马尿苷和马尿苷依赖性方式增强甲硫氨酰嘌呤霉素合成。 eIF5A 与主动翻译的核糖体结合，eIF5A 的条件突变体对蛋白质合成抑制剂高度敏感。我们使用酵母菌株 UBHY-R（源自 John WB Hershey 博士的实验室）研究了 eIF5A 在翻译中的作用。在该菌株中，两个酵母 eIF5A 基因失活，其生长依赖于基因工程改造的不稳定融合 eIF5A (UBR5A)，在 GAL 启动子下表达。当将该菌株从半乳糖培养基 (YPGal) 转移到葡萄糖培养基 (YPD) 时，UBR5A 迅速降解，蛋白质合成下降，细胞生长受到抑制。 UBHY-R 菌株中人野生型 eIF5A 的表达恢复了蛋白质合成和生长，而失活的突变型 eIF5A 蛋白的表达则没有影响。从酵母温度敏感的 eIF5A 突变体中获得了 eIF5A 在翻译中直接作用的进一步证据。两种温度敏感酵母菌株产生稳定的突变蛋白，eIF5AK56A 和 eIF5AQ22H,L93F。当这两种菌株转移到限制性温度时，观察到蛋白质合成的快速抑制，这可能是由于温度转移时突变蛋白构象改变的结果。