eIF4E作用及调节机制

eIF4E作为翻译启动因子复合体eIF4-F的关键部分,调节细胞的蛋白质合成的限速步骤,ras基因加强eIF4E的磷酸化,c-myc基因增加eIF4E mRNA水平,eIF4E活性随其本身磷酸化的加强而加强,随其结合蛋白的磷酸化加强而减弱。eIF4E是新近发现的原癌,这量的eIF4E选择性地加强原癌基因等“弱mRNA”的翻译：可以通过加强蛋白质合成的速度,也可以加速mRNA由细胞核由胞浆转移。     

应用套叠PCR技术构建番木瓜eIF4E和eIFiso4E基因的hpRNA植物表达载体

植物真核翻译起始因子4E(eIF4E)在蛋白质合成的起始中发挥重要作用,参与植物-病毒互作,影响病毒的侵染过程.为了研究eIF4E在植物病毒侵染中的功能,建立了一种快速的套叠PCR新方法,成功构建了番木瓜eIF4E和eIFiso4E基因的hpRNA结构,并将其连接到改造的植物表达载体pBI121上,为利用RNA干扰技术研究番木瓜eIF4E和eIFiso4E基因在病毒侵染中的作用奠定了基础.     

本生烟eIF4Es基因克隆及其酵母双杂交诱饵载体构建

由木薯褐色条斑病毒(Cassava brown steak virus, CBSV)和乌干达木薯褐色条斑病毒(Uganda Cassava brown steak virus, UCBSV)引起的木薯褐色条斑病毒病,是危害木薯的主要病害之一。目前尚无有效防治方法,并且特别是缺乏CBSV/UCBSV抗性育种材料。真核翻译起始因子4E (eukaryotie translation initiation factor4E, eIF4E)不仅参与蛋白质的翻译起始,并且Potyviruses的复制和翻译也依赖于eIF4E与病毒基因组连接蛋白(virus genome linked protein, VPg)的相互作用,目前发现的植物隐性抗病基因大部分都是eIF4E家族的等位基因。本研究利用生物信息学方法和RT-PCR技术获得本生烟(Nicotiana benthamiana) eIF4E家族的8个基因,聚类分析显示它们分别编码eIF4E、eIF4E的异构体和类似帽结合的蛋白。为筛选与CBSV/UCBSV相互作用的本生烟eIF4E家族蛋白,本研究进一步构建了这8个基因的Lex A-酵母双杂交系统的诱饵载体,并导入酵母细胞EGY48 (p8op-LacZ)进行细胞毒性和自激活检测。结果显示导入重组质粒的EGY48 (p8op-LacZ)酵母细胞在SD/-His/-Ura缺陷型培养基上生长良好,在SD/-Trp/-Ura缺陷型平板上不生长,并且在SD/Gal/Raf/-His/-Ura/X-Gal培养基上不显蓝色,说明重组质粒表达产物不仅对该酵母细胞无毒性,而且对下游报告基因无自激活作用,可用于该系统的互作蛋白筛选研究。本研究为利用酵母双杂交系统发现与CBSV/UCBSV相互作用的本生烟eIF4E基因,探索通过基因编辑与CBSV/UCBSV互作的寄主因子进行抗CBSV/UCBSV分子育种提供科学依据。     

番木瓜eIF4E和eIFiso4E基因嵌合hpRNA载体一步快速构建及沉默效果的研究

番木瓜环斑病毒(Papaya ringspot virus,PRSV)严重威胁番木瓜种植业的发展,且目前没有十分有效的防治办法。病毒侵染植物依赖寄主因子的协助,真核翻译起始因子4E(eukaryotic initiation factor 4E,eIF4E)是多种RNA病毒侵染植物的必需因子。以番木瓜eIF4E家族基因为研究对象,构建同时干扰其eIF4E和eIFiso4E基因的发卡RNA(hairpin RNA,hpRNA)载体,并将其导入到番木瓜叶肉原生质中。通过荧光实时定量检测发现,番木瓜中eIF4E和eIFiso4E基因的表达量分别下降了49.8%和67.6%,这为进一步研究番木瓜eIF4E家族基因对PRSV侵染的影响以及利用RNA干扰技术靶向植物基因的病毒防治新策略提供理论和实践依据。     

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。     

NDV诱导HeLa细胞发生核糖体应激及对翻译起始复合体的影响

[目的]研究新城疫病毒(Newcastle disease virus,NDV) HBUN/LSRC/F3株(以下简称NDV F3)诱导宫颈癌细胞(HeLa)发生核糖体应激后对eIF2α介导的翻译起始复合体eIF4F的调控作用。[方法]流式细胞术及CCK-8检测细胞凋亡;实时荧光定量PCR (quantitative real-time polymerase chain reaction,qRT-PCR)检测c-Myc基因表达;流式细胞术分析细胞周期;Western blotting技术检测c-Myc、RPS7、Bcl-2、NP、eIF4E及eIF2α蛋白的表达;Western blotting和免疫荧光染色技术检测NP、eIF4E蛋白定位。[结果]与阴性对照组相比,NDV F3抑制HeLa细胞增殖并诱导细胞凋亡。细胞周期中G₀/G₁期出现停滞,c-Myc表达呈时间依赖性抑制,c-Myc与Bcl-2蛋白表达量在0-48 h内逐渐下降,NP蛋白在24 h时生成并逐渐增加,RPS7、eIF4E和eIF2α蛋白含量在0-48 h内呈先增加后降低趋势。Western blotting定位分析及激光共聚焦显微镜结果显示NP蛋白主要存在细胞质中,NP与eIF4E存在共定位现象。[结论]NDV F3诱导HeLa细胞凋亡并引发核糖体应激反应,NP与eIF4E相互作用而抑制eIF2α介导的翻译起始复合体eIF4F形成,阻断其与宿主mRNA之间的联系,同时促进NDV F3 mRNA的翻译,最终造成宿主蛋白翻译抑制。     

eIF4A1与TrkA相互作用后抑制TrkA的泛素化

神经生长因子（NGF）结合细胞表面受体p75NTR(p75神经营养素受体)和TrkA（酪氨酸蛋白激酶A）后介导了细胞分化、细胞生存、凋亡、增殖和侵袭等多个重要的生理病理过程. TrKA能与细胞内多个蛋白质相互作用,但是由于NGF信号通路的复杂性,现在仍有必要发现与之相互作用的蛋白质以更准确地了解NGF信号通路. 本研究中我们通过酵母双杂交的方法筛选到了一个新的与TrKA相互作用的蛋白质——真核生物翻译起始因子4A1（eIF4A1）,然后通过谷胱甘肽巯基转移酶融合蛋白沉降实验（GST-pull-down）和免疫共沉淀实验（Co-IP）证明了TrkA和eIF4A1的相互作用. 此外NGF能够增强TrkA和eIF4A1的相互作用. 在鉴定相互作用位点实验中,我们发现eIF4A1的氨基端结构域和TrkA的TK结构域参与了相互作用. TrkA和eIF4A1共定位在细胞膜上. NGF能够引起TrkA与泛素蛋白63位的赖氨酸连接,而eIF4A1与TrkA相互作用后能够抑制TrkA与泛素蛋白63位的赖氨酸连接. 综上,得出结论 eIF4A1通过与TrkA相互作用抑制其泛素化调控NGF信号通路.     

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。     

我国辣椒种质资源eIF4E基因多态性分析

利用国家蔬菜种质资源库的1904份辣椒资源材料,采用测序技术获得eIF4E(eukaryotic translation initiation 4E)基因exon1序列,研究e IF4E基因多样性及我国辣椒种质资源群体多样性。结果表明:在1904份材料中共发现17个单倍型,14个有义多态性位点,其中9个为新的位点,位点大多集中在eIF4E蛋白表面环上;8个地理群体的平均单倍型多样性(Hd)和平均核苷酸多样性(Pi)分别为0.519和0.00210;群体间分化指数(Fst值)及基因流(Nm)表明不同群体间表现差异的分化程度;AMOVA分析表明总变异主要来源于群体内个体间的变异(97.23%),只有2.77%变异发生在群体间。本研究将有助于了解我国辣椒eIF4E基因多样性,为抗PVY育种提供更多抗源材料。     

水稻eIF3大亚基（eIF3a）编码基因的克隆及其表达模式分析

真核生物翻译起始因子（eIFs)在蛋白合成中起关键作用,在已经鉴定 的13个因子中eIF3（由8个或更多的亚基组成）是分子量最大的一个并在翻译起始过程中起着核心作用。eIF3a是eIF3中最大的亚基并介导了eIF3的大多数的功能的实现。利用荧光－差异显示PCR法对生长素处理后的水稻材料进行分析发现eIF3a的表达可被生长素诱导,进一步通过筛选cDNA文库分离出水稻编码eIF3a的全长cDNA（命名为OseIF3a1),OseIF3a1含3459碱基（含5‘和3‘非翻译区）并编码一个986氨基酸的蛋白,与基因组序列比较表明OseIF3a1基因存在有12个内含子。OseIF3a1与玉米和烟草的同源蛋白一致性分别为82．4％和70．1％并与其他真核生物eIF3a蛋白具较高一致性。以组织材料进行的RT－PCR结果表明OseIF3a1在根、幼苗、幼穗、茎和叶中表达,启动子－报告基因的转基因结果进一步表明OseIF3a1在根尖及幼嫩叶片表达较高,进一步的RT－PCR分析确证了生长素对OseIF3a1的诱导,表明生长素在调控植物生长时可能涉及了翻译水平上的调节。     

Suppression of translation during in vitro maturation of pig oocytes despite enhanced formation of cap-binding protein complex eIF4F and 4E-BP1 hyperphosphorylation

In this study, we document that the overall rate of protein synthesis decreases during in vitro maturation (IVM) of pig oocytes despite enhanced formation of the 5' cap structure eIF4F. Within somatic/interphase cells, formation of the eIF4F protein complex correlates very well with overall rates of protein translation, and the formation of this complex is controlled primarily by the availability of the 5' cap binding protein eIF4E. We show that the eIF4E inhibitory protein, 4E-BP1, becomes phosphorylated during IVM, which results in gradual release of eIF4E from 4E-BP1, as documented by immunoprecipitation analyses. Isoelectric focusing and Western blotting experiments show conclusively that eIF4E becomes gradually phosphorylated with a maximum at metaphase II (M II). The activity of eIF4E and its ability to bind mRNA also increases during oocyte maturation as documented in experiments with m7-methyl GTP-Sepharose, which mimics the cap structure of mRNA. Complementary analysis of flow-through fraction for 4E-BP1, and eIF4G proteins additionally provides evidence for enhanced formation of cap-binding protein complex eIF4F. Altogether, our results bring new insights to the regulation of translation initiation during meiotic division, and more specifically clarify that 4E-BP1 hyper-phosphorylation is not the cause of the observed suppression of overall translation rates.     

Activation of p53 stimulates proteasome-dependent truncation of eIF4E-binding protein 1 (4E-BP1)

Background information. The translational inhibitor protein 4E‐BP1 [eIF4E (eukaryotic initiation factor 4E)‐binding protein 1] regulates the availability of polypeptide chain initiation factor eIF4E for protein synthesis. Initiation factor eIF4E binds the 5′ cap structure present on all cellular mRNAs. Its ability to associate with initiation factors eIF4G and eIF4A, forming the eIF4F complex, brings the mRNA to the 43S complex during the initiation of translation. Binding of eIF4E to eIF4G is inhibited in a competitive manner by 4E‐BP1. Phosphorylation of 4E‐BP1 decreases the affinity of this protein for eIF4E, thus favouring the binding of eIF4G and enhancing translation. We have previously shown that induction or activation of the tumour suppressor protein p53 rapidly leads to 4E‐BP1 dephosphorylation, resulting in sequestration of eIF4E, decreased formation of the eIF4F complex and inhibition of protein synthesis. Results. We now report that activation of p53 also results in modification of 4E‐BP1 to a truncated form. Unlike full‐length 4E‐BP1, which is reversibly phosphorylated at multiple sites, the truncated protein is almost completely unphosphorylated. Moreover, the latter interacts with eIF4E in preference to full‐length 4E‐BP1. Inhibitor studies indicate that the p53‐induced cleavage of 4E‐BP1 is mediated by the proteasome and is blocked by conditions that inhibit the dephosphorylation of full‐length 4E‐BP1. Measurements of the turnover of 4E‐BP1 indicate that the truncated form is much more stable than the full‐length protein. Conclusions. The results suggest a model in which proteasome activity gives rise to a stable, hypophosphorylated and truncated form of 4E‐BP1, which may exert a long‐term inhibitory effect on the availability of eIF4E, thus contributing to the inhibition of protein synthesis and the growth‐inhibitory and pro‐apoptotic effects of p53.     

Sumoylation of eIF4E activates mRNA translation

Eukaryotic translation initiation factor 4E (eIF4E) is the cap‐binding protein that binds the 5′ cap structure of cellular messenger RNAs (mRNAs). Despite the obligatory role of eIF4E in cap‐dependent mRNA translation, how the translation activity of eIF4E is controlled remains largely undefined. Here, we report that mammalian eIF4E is regulated by SUMO1 (small ubiquitin‐related modifier 1) conjugation. eIF4E sumoylation promotes the formation of the active eIF4F translation initiation complex and induces the translation of a subset of proteins that are essential for cell proliferation and preventing apoptosis. Furthermore, disruption of eIF4E sumoylation inhibits eIF4E‐dependent protein translation and abrogates the oncogenic and antiapoptotic functions associated with eIF4E. These data indicate that sumoylation is a new fundamental regulatory mechanism of protein synthesis. Our findings suggest further that eIF4E sumoylation might be important in promoting human cancers.     

Coordinated and selective recruitment of eIF4E and eIF4G factors for potyvirus infection in Arabidopsis thaliana

The translation initiation factors eIF4E and eIF(iso)4E play a key role during virus infection in plants. During mRNA translation, eIF4E provides the cap-binding function and is associated with the protein eIF4G to form the eIF4F complex. Susceptibility analyses of Arabidopsis mutants knocked-out for At-eIF4G genes showed that eIF4G factors are indispensable for potyvirus infection. The colonization pattern by a viral recombinant carrying GFP indicated that eIF4G is involved at a very early infection step. Like eIF4E, eIF4G isoforms are selectively recruited for infection. Moreover, the eIF4G selective involvement parallels eIF4E recruitment. This is the first report of a coordinated and selective recruitment of eIF4E and eIF4G factors, suggesting the whole eIF4F recruitment.     

Eukaryotic initiation factor 4E

Eukaryotic translation initiation factor 4E (eIF4E) is perhaps best known for its function in the initiation of protein synthesis on capped mRNAs in the cytoplasm. However, recent studies have highlighted that eIF4E has many additional functions, which include the nuclear export of specific mRNAs as well as roles in ageing and the translation of some uncapped viral RNAs. This review aims to update the reader on recent developments, including the potential of eIF4E as a therapeutic target.     

Structure and functions of the translation initiation factor eIF4E and its role in cancer development and treatment

In eukaryotic cells, protein synthesis is a complex and multi-step process that has several mechanisms to start the translation including cap-dependent and cap-independent initiation. The translation control of eukaryotic gene expression occurs principally at the initiation step. In this context, it is critical that the eukaryotic translation initiation factor eIF4E bind to the 7-methylguanosine (m7G) cap present at the 5′-UTRs of most eukaryotic mRNAs. Combined with other initiation factors, eIF4E mediates the mRNA recruitment on ribosomes to start the translation. Moreover, the eIF4E nuclear bodies are involved in the export of specific mRNAs from the nucleus to the cytoplasm. In this review, we focus on the eIF4E structure and its physiological functions, and describe the role of eIF4E in cancer development and progression and the current therapeutic strategies to target eIF4E.     

eIF4E/4E-BP dissociation and 4E-BP degradation in the first mitotic division of the sea urchin embryo

The mRNA's cap-binding protein eukaryotic translation initiation factor (eIF)4E is a major target for the regulation of translation initiation. eIF4E activity is controlled by a family of translation inhibitors, the eIF4E-binding proteins (4E-BPs). We have previously shown that a rapid dissociation of 4E-BP from eIF4E is related with the dramatic rise in protein synthesis that occurs following sea urchin fertilization. Here, we demonstrate that 4E-BP is destroyed shortly following fertilization and that 4E-BP degradation is sensitive to rapamycin, suggesting that proteolysis could be a novel means of regulating 4E-BP function. We also show that eIF4E/4E-BP dissociation following fertilization is sensitive to rapamycin. Furthermore, while rapamycin modestly affects global translation rates, the drug strongly inhibits cyclin B de novo synthesis and, consequently, precludes the completion of the first mitotic cleavage. These results demonstrate that, following sea urchin fertilization, cyclin B translation, and thus the onset of mitosis, are regulated by a rapamycin-sensitive pathway. These processes are effected at least in part through eIF4E/4E-BP complex dissociation and 4E-BP degradation.     

Regulation of Eukaryotic Initiation Factor 4E and Its Isoform： Implications for Antiviral Strategy in Plants

In recent years, biotechnology has permitted regulation of the expression of endogenous plant genes to improve agronomlcally important traits. Genetic modification of crops has benefited from emerging knowledge of new genes, especially genes that exhibit novel functions, one of which is eukaryotlc initiation factor 4E （eIF4E）. eIF4E Is one of the most important translation initiation factors Involved in eukaryotic initiation. Recent research has demonstrated that virus resistance mediated by eIF4E and Its isoform elf （Iso）4E occurs in several plant-virus interactions, thus indicating a potential new role for eIF4E/elF（Iso）4E In resistance strategies against plant viruses. In this review, we briefly describe eIF4E activity In plant translation, its potential role, and functions of the eIF4E subfamily In plant-virus interactions. Other initiation factors such as elF4G could also play a role In plant resistance against viruses. Finally, the potential for developing eIF4E-mediated resistance to plant viruses in the future Is discussed. Future research should focus on elucidation of the resistance mechanism and spectrum mediated by eIF4E. Knowledge of a particu- lar plant-virus interaction will help to deepen our understanding of eIF4E and other eukaryotic Initiation factors, and their involvement in virus disease control.     

Potyvirus terminal protein VPg, effector of host eukaryotic initiation factor eIF4E

Potyvirus RNA contains at the 5' end a covalently linked virus-encoded protein VPg, which is required for virus infectivity. This role has been attributed to VPg interaction with the eukaryotic translation initiation factor eIF4E, a cap-binding protein. We characterized the dissociation constants for the interaction of the potato virus Y VPg with different plant eIF4Es and its isoforms and mapped the eIF(iso)4E attachment region on VPg. VPg/eIF4E interaction results in the inhibition of cell-free protein synthesis, and we show that it stems from the liberation of the cap moiety from the complex with eIF4E. Since VPg does not attach the cap, it appears that VPg induces changes in the eIF4E structure, diminishing its affinity to the cap. We show here that the initiation complex scaffold protein eIF(iso)4G increases VPg interaction with eIF(iso)4E. These data together suggest similar cap and VPg interactions with eIF4E and characterize VPg as a novel eIF4E-binding protein, which inhibits host protein synthesis at a very early stage of the initiation complex formation through the inhibition of cap attachment to the initiation factor eIF4E.     

Stabilizing the eIF4G1 α-helix increases its binding affinity with eIF4E: implications for peptidomimetic design strategies

Eukaryotic initiation factor (eIF)4E is overexpressed in many types of cancer such as breast, head and neck, and lung. A consequence of increased levels of eIF4E is the preferential translation of pro-tumorigenic proteins such as c-Myc, cyclin D1, and vascular endothelial growth factor. Inhibition of eIF4E is therefore a potential therapeutic target for human cancers. A novel peptide based on the eIF4E-binding peptide eIF4G1, where the α-helix was stabilized by the inclusion of α-helix inducers as shown by CD measurements, was synthesized. The helically stabilized peptide binds with an apparent K_d of 9.43 ± 2.57 nM, which is ∼ 15.7-fold more potent than the template peptide from which it is designed. The helically stabilized peptide showed significant biological activity at a concentration of 400 μM, unlike the naturally occurring eIFG1 peptide when measured in cell-based cap-dependent translational reporter and WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assays. Fusion of the template peptide and the stabilized peptide to the cell-penetrating peptide TAT produced more active but equally potent inhibitors of cap-dependent translation in cell lines. They also equally disrupted cell metabolism as measured in a WST-1 assay. Propidium iodide staining revealed that the TAT-fused, helically stabilized peptide caused more cell death than the TAT-fused eIF4G1 template peptide with substantial decreases in the G1 and G2 cell populations. Annexin-staining experiments also indicated that the TAT-fused eIF4G1 derivative peptides caused cell death by apoptosis. The results presented should offer further insight into peptidomimetics development for eIF4E.