转eIF -5A及其修饰酶DHS基因选育作物新品种的研究进展

概述eIF -5A和DHS的研究与应用的进展.脱羧腐胺赖氨酸合酶(deoxyhypusine synthase,DHS)广泛分布在微生物、植物、动物的细胞中,同时参与真核细胞翻译起始因子eIF - 5A( eukaryotic initiation factor - 5A)的蛋白质合成及其延伸与折叠和翻译后修饰功能.可见eIF - 5A具有多种生物学功能,主要参与蛋白质翻译、合成蛋白质的延伸和折叠、细胞增殖、细胞周期的转化、mRNA的运输、降解、细胞衰老与凋亡等.而且eIF - 5A是DHS的唯一底物,DHS的表达被抑制的植物表现为速生、抗逆性增强、种子的产量和植株增高,开花期与果实储存期延长的特征.转调控DHS和eIF - 5A基因可能是一种农作物遗传育种的新途径.     

eIF4A在帽依赖和IRES介导的翻译起始中的作用

生物体内翻译起始机制分为两类：帽依赖性翻译起始和内部核糖体进入位点(internal ribosome entry sites, IRES)介导的翻译起始。真核生物的翻译起始为经典的帽依赖性翻译起始模型,而大多数正链RNA病毒选择依赖于IRES的翻译机制。真核翻译起始因子4A (eukaryotic initiation factor 4A, eIF4A)是DEAD-box RNA解旋酶家族的成员,具有依赖于RNA的ATP酶活性和RNA解旋酶活性,而e IF4A具体的解旋机制至今仍不清晰。同时,eIF4A与其他翻译因子有着复杂而紧密的联系,在帽依赖性与IRES介导的翻译起始过程中扮演着至关重要的角色。本文主要对eIF4A的功能、结构以及eIF4A在帽依赖性与IRES介导的翻译起始过程中的机制作一综述。     

eIF3a与肿瘤发生、演进和干预的研究进展

真核翻译起始因子3(Eukaryotic translation factor 3,eIF3)是由多个亚单位组成的复合因子,其中eIF3a是其最大的亚单位。很多研究表明在酵母和哺乳动物细胞中,eIF3都参与了m RNA翻译起始,并对蛋白质的合成有很好的调控作用。值得一提的是eIF3a通过调控一系列与肿瘤的生成、细胞周期的调控DNA修复等过程相关的m RNA的翻译从而在肿瘤的发生、演进和干预中发挥重要作用。此外,研究发现eIF3a对RAF-MEK-ERK信号通路有抑制作用。eIF3a对蛋白质翻译的调节及其对RAF-MEK-ERK信号通路的影响使其有望成为肿瘤治疗的新靶点。本文将着重围绕eIF3a在肿瘤发生、演进和干预中的作用进行概述。     

蛋白质生物合成的第四步：翻译终止后复合物的解体

蛋白质合成过程一般被归纳为由合成的起始、肽链的延伸和合成的终止组成的三步曲 . 然而,随着对核糖体再循环因子 (ribosome recycling factor , RRF) 在蛋白质合成过程中作用的深入研究,人们提出了蛋白质生物合成应是四步曲, 这第四步就是翻译终止后核糖体复合物的解体 , 也就是通常说的核糖体循环再利用 . 简要地介绍了翻译终止后复合物解体的可能机制：核糖体再循环因子和蛋白质合成延伸因子 G 在核糖体上协同作用催化这一过程的完成 .     

真核翻译起始因子5A和多胺代谢及肿瘤的关系

真核翻译起始因子5A(eIF5A)是真核生物中普遍存在且高度保守的蛋白质,含有一个独特的氨基酸——羟腐胺赖氨酸,该氨基酸是在赖氨酸的基础上经翻译后修饰而成,该过程与多胺代谢密切相关。研究发现,eIF5A和多胺代谢以及肿瘤的发生发展密切相关,有望成为抗肿瘤靶向治疗新的分子靶点。现就eIF5A和多胺代谢及肿瘤关系的研究进展作一简要综述。     

真核生物翻译起始机制

蛋白质生物合成是遗传信息的翻译过程,是基因表达的第二个阶段,整个翻译包括起始、延伸和终止3个阶段。其中起始阶段最为复杂,是调控的关键。在真核生物中,在各种起始因子的参与下,通过蛋白一蛋白和蛋白HNA的相互作用,使405核糖体小亚基（预起始复合物）与mRNA相互作用,形成起始复合物,再与6OS大亚基相结合。蛋白质合成起始,形成肽健,从而进入延伸阶段关于起始作用的机理关键在于4OS／J‘亚基富集（recruit）于mRNA的过程。即核糖体是如何鉴别mRNA上的起始密码子（AUG）,以适当的阅读框架开始翻译的。结合的方式目前有两…     

保守GTP酶EF-G在蛋白质合成中的功能研究北大核心CSCD

延伸因子G(elongation factor G,EF-G)是一种保守的GTP水解酶,它是蛋白质翻译过程中一个重要的调控因子。同源模拟发现EF-G与核糖体保护蛋白Tet(O)具有相似的空间结构且都包含5个结构域,序列比对发现E.coliEF-G与Campylobacter jejuniTet(O)结构域Ⅳ保守的两个环状区不同。通过分子克隆构建EF-G嵌合体,表达纯化后的蛋白突变体通过核糖体依赖的GTP水解酶(GTPase)活性检测、多聚尿嘧啶(polyU)为mRNA合成苯丙氨酸多肽链、多聚核糖体的解聚检测及相关的体内实验,检测EF-G在肽链合成中的作用,结果发现EF-G嵌合体能够影响肽链生成过程中tRNA-mRNA复合物的移位,但不影响核糖体的再循环过程。     

真核翻译因子与蛋白质生物合成

真核mRNA在80S核糖体上翻译成蛋白质是一个复杂的过程,需要多步反应及多种因子参与,文章就真核蛋白质的生物合成机制简要综述翻译起始、延伸和终止因子的结构、功能和性质及其在肽链合成过程中的作用研究新进展.     

丙型肝炎病毒5

丙型肝炎病毒(HCV)RNA5′-非编码区(5′-NTR)由341个核苷酸组成,形成4个茎-环二级结构,5′-NTR二级结构及某些部分单链序列的核苷酸组成是病毒翻译起始的先决条件.5′-NTR中的大部分核苷酸序列组成内部核糖体进入位点(IRES),在宿主细胞蛋白质因子La自身抗原、eIF3、多聚嘧啶区结构蛋白(PTB)、多聚胞嘧啶结合蛋白(PCBP-1、2)等的作用下,形成复杂的翻译起始复合物,对HCV的翻译过程进行精确调控,完成帽状结构形成非依赖性的蛋白翻译过程.HCV RNA 5′-NTR翻译过程的分子生物学机制的研究,将有助于HCV治疗新方法和新途径的探索.     

丙型肝炎病毒5′-非编码区及其结合蛋白的研究进展

丙型肝炎病毒(HCV)RNA5′-非编码区(5′-NTR)由341个核苷酸组成,形成4个茎-环二级结构,5′-NTR二级结构及某些部分单链序列的核苷酸组成是病毒翻译起始的先决条件.5′-NTR中的大部分核苷酸序列组成内部核糖体进入位点(IRES),在宿主细胞蛋白质因子La自身抗原、eIF3、多聚嘧啶区结构蛋白(PTB)、多聚胞嘧啶结合蛋白(PCBP-1、2)等的作用下,形成复杂的翻译起始复合物,对HCV的翻译过程进行精确调控,完成帽状结构形成非依赖性的蛋白翻译过程.HCV RNA 5′-NTR翻译过程的分子生物学机制的研究,将有助于HCV治疗新方法和新途径的探索.     

The effect of hypusine modification on the intracellular localization of eIF5A

Eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein essential for eukaryotic cell proliferation and is the only protein containing hypusine, [N^ε-(4-amino-2-hydroxybutyl)lysine]. eIF5A is activated by the post-translational synthesis of hypusine. eIF5A also undergoes an acetylation at specific Lys residue(s). In this study, we have investigated the effect of hypusine modification and acetylation on the subcellular localization of eIF5A. Immunocytochemical analyses showed differences in the distribution of non-hypusinated eIF5A precursor and the hypusine-containing mature eIF5A. While the precursor is found in both cytoplasm and nucleus, the hypusinated eIF5A is primarily localized in cytoplasm. eIF5A mutant proteins, defective in hypusine modification (K50A, K50R) were localized in a similar manner to the eIF5A precursor, whereas hypusine-modified mutant proteins (K47A, K47R, K68A) were localized mainly in the cytoplasm. These findings provide strong evidence that the hypusine modification of eIF5A dictates its localization in the cytoplasmic compartment where it is required for protein synthesis.     

Molecular modeling of the human eukaryotic translation initiation factor 5A (eIF5A) based on spectroscopic and computational analyses

The eukaryotic translation initiation factor 5A (eIF5A) is a protein ubiquitously present in archaea and eukarya, which undergoes a unique two-step post-translational modification called hypusination. Several studies have shown that hypusination is essential for a variety of functional roles for eIF5A, including cell proliferation and synthesis of proteins involved in cell cycle control. Up to now neither a totally selective inhibitor of hypusination nor an inhibitor capable of directly binding to eIF5A has been reported in the literature. The discovery of such an inhibitor might be achieved by computer-aided drug design based on the 3D structure of the human eIF5A. In this study, we present a molecular model for the human eIF5A protein based on the crystal structure of the eIF5A from Leishmania brasiliensis, and compare the modeled conformation of the loop bearing the hypusination site with circular dichroism data obtained with a synthetic peptide of this loop. Furthermore, analysis of amino acid variability between different human eIF5A isoforms revealed peculiar structural characteristics that are of functional relevance.     

The N-terminal region of eukaryotic translation initiation factor 5A signals to nuclear localization of the protein

The eukaryotic translation initiation factor 5A (eIF5A) is a ubiquitous protein of eukaryotic and archaeal organisms which undergoes hypusination, a unique post-translational modification. We have generated a polyclonal antibody against murine eIF5A, which in immunocytochemical assays in B16-F10 cells revealed that the endogenous protein is preferentially localized to the nuclear region. We therefore analyzed possible structural features present in eIF5A proteins that could be responsible for that characteristic. Multiple sequence alignment analysis of eIF5A proteins from different eukaryotic and archaeal organisms showed that the former sequences have an extended N-terminal segment. We have then performed in silico prediction analyses and constructed different truncated forms of murine eIF5A to verify any possible role that the N-terminal extension might have in determining the subcellular localization of the eIF5A in eukaryotic organisms. Our results indicate that the N-terminal extension of the eukaryotic eIF5A contributes in signaling this protein to nuclear localization, despite of bearing no structural similarity with classical nuclear localization signals.     

eIF5A has a function in the elongation step of translation in yeast

The putative translation factor eIF5A is essential for cell viability and is highly conserved throughout evolution. Here, we describe genetic interactions between an eIF5A mutant and a translation initiation mutant (eIF4E) or a translation elongation mutant (eEF2). Polysome profile analysis of single and double mutants revealed that mutation in eIF5A reduces polysome run-off, contrarily to translation initiation mutants. Moreover, the polysome profile of an eIF5A mutant alone is very similar to that of a translation elongation mutant. Furthermore, depletion of eIF5A causes a significant decrease in total protein synthesis and an increase of the average ribosome transit time. Finally, we demonstrate that the formation of P bodies is inhibited in an eIF5A mutant, similarly to the effect of the translation elongation inhibitor cycloheximide. Taken together, these results not only reinforce a role for eIF5A in translation but also strongly support a function for eIF5A in the elongation step of protein synthesis.     

Inhibition of eukaryotic translation initiation factor 5A (eIF5A) hypusination impairs melanoma growth

The eukaryotic translation initiation factor 5A (eIF5A) undergoes a specific post-translational modification called hypusination. This modification is required for the functionality of this protein. The compound N1-guanyl-1,7-diaminoheptane (GC7) is a potent and selective inhibitor of deoxyhypusine synthase, which catalyses the first step of eIF5A hypusination process. In the present study, the effects of GC7 on cell death were investigated using two cell lines: melan-a murine melanocytes and Tm5 murine melanoma. In vitro treatment with GC7 increased by 3-fold the number of cells presenting DNA fragmentation in Tm5 cells. Exposure to GC7 also decreased viability to both cell lines. This study also describes, for the first time, the in vivo antitumour effect of GC7, as indicated by impaired melanoma growth in C57BL/6 mice.     

Deoxyhypusine Modification of Eukaryotic Translation Initiation Factor 5A (eIF5A) Is Essential for Trypanosoma brucei Growth and for Expression of Polyprolyl-containing Proteins

The eukaryotic protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis. Polyamine biosynthesis is essential in T. brucei, and the polyamine spermidine is required for synthesis of a novel cofactor called trypanothione and for deoxyhypusine modification of eukaryotic translation initiation factor 5A (eIF5A). eIF5A promotes translation of proteins containing polyprolyl tracts in mammals and yeast. To evaluate the function of eIF5A in T. brucei, we used RNA interference (RNAi) to knock down eIF5A levels and found that it is essential for T. brucei growth. The RNAi-induced growth defect was complemented by expression of wild-type human eIF5A but not by a Lys-50 mutant that blocks modification by deoxyhypusine. Bioinformatics analysis showed that 15% of the T. brucei proteome contains 3 or more consecutive prolines and that actin-related proteins and cysteine proteases were highly enriched in the group. Steady-state protein levels of representative proteins containing 9 consecutive prolines that are involved in actin assembly (formin and CAP/Srv2p) were significantly reduced by knockdown of eIF5A. Several T. brucei polyprolyl proteins are involved in flagellar assembly. Knockdown of TbeIF5A led to abnormal cell morphologies and detached flagella, suggesting that eIF5A is important for translation of proteins needed for these processes. Potential specialized functions for eIF5A in T. brucei in translation of variable surface glycoproteins were also uncovered. Inhibitors of deoxyhypusination would be expected to cause a pleomorphic effect on multiple cell processes, suggesting that deoxyhypusine/hypusine biosynthesis could be a promising drug target in not just T. brucei but in other eukaryotic pathogens.     

rRNA Suppressor of a Eukaryotic Translation Initiation Factor 5B/Initiation Factor 2 Mutant Reveals a Binding Site for Translational GTPases on the Small Ribosomal Subunit

The translational GTPases promote initiation, elongation, and termination of protein synthesis by interacting with the ribosome. Mutations that impair GTP hydrolysis by eukaryotic translation initiation factor 5B/initiation factor 2 (eIF5B/IF2) impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. A mutation in helix h5 of the 18S rRNA in the 40S ribosomal subunit and intragenic mutations in domain II of eIF5B suppress the toxic effects associated with expression of the eIF5B-H480I GTPase-deficient mutant in yeast by lowering the ribosome binding affinity of eIF5B. Hydroxyl radical mapping experiments reveal that the domain II suppressors interface with the body of the 40S subunit in the vicinity of helix h5. As the helix h5 mutation also impairs elongation factor function, the rRNA and eIF5B suppressor mutations provide in vivo evidence supporting a functionally important docking of domain II of the translational GTPases on the body of the small ribosomal subunit.     

An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation

In vitro translation systems are used to investigate translational mechanisms and to synthesize proteins for characterization. Most available mammalian cell-free systems have reduced efficiency due to decreased translation initiation caused by phosphorylation of the initiation factor eIF2alpha on Ser51. We describe here a novel cell-free protein synthesis system using extracts from cultured mouse embryonic fibroblasts that are homozygous for the Ser51 to- Ala mutation in eIF2alpha (A/A cells). The translation efficiency of a capped and polyadenylated firefly luciferase mRNA in A/A cell extracts was 30-fold higher than in wild-type extracts. Protein synthesis in extracts from A/A cells was active for at least 2 h and generated up to 20 microg/mL of luciferase protein. Additionally, the A/A cell-free system faithfully recapitulated the selectivity of in vivo translation for mRNA features; translation was stimulated by a 5'-end cap (m7GpppN) and a 3'-end poly(A) tail in a synergistic manner. The system also showed similar efficiencies of cap-dependent and IRES-mediated translation (EMCV IRES). Significantly, the A/A cell-free system supported the post-translational modification of proteins, as shown by glycosylation of the HIV type-1 gp120 and cleavage of the signal peptide from beta-lactamase. We propose that cell-free systems from A/A cells can be a useful tool for investigating mechanisms of mammalian mRNA translation and for the production of recombinant proteins for molecular studies. In addition, cell-free systems from differentiated cells with the Ser51Ala mutation should provide a means for investigating cell type-specific features of protein synthesis.