真核生物蛋白质翻译的内部起始机制

蛋白质翻译过程中,翻译的起始步骤是非常重要的．真核生物的翻译起始主要是通过依赖帽子结构的扫描机制进行的．近几年在翻译的研究工作中发现,在一些动物病毒中,蛋白质合成通过一种不同于扫描机制的内部起始机制起始翻译．用内部起始机制翻译的mRNA的5′端非翻译区有一个相对保守的结构,它在内部起始过程中具有重要作用,一些特异的蛋白质因子能够促进在特定位点起始翻译．     

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。     

翻译起始因子5A2与肿瘤的研究进展

真核翻译起始因子5A2(eukaryotic translation initiation factor 5A2,e IF5A2)是一种在真核细胞蛋白质翻译起始和延伸过程中发挥作用的蛋白,它是e IF5A的其中一个亚型,在多种肿瘤细胞中的异常高表达往往与该肿瘤的发生和发展相关。e IF5A2所特有的羟腐赖氨酸,可以成为肿瘤治疗的潜在靶点,为临床带来新的思路。     

真核翻译起始因子3与肿瘤

真核翻译起始因子3是由多个亚基组成的,在真核翻译起始中发挥重要作用,近年来的研究表明其多个亚基在多种肿瘤细胞中存在异常表达的现象且与肿瘤的侵袭性、转移能力、分化程度及预后相关,使其有望成为肿瘤治疗的新靶点。     

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…     

RNA病毒翻译调控元件—内部核糖体进入位点(IRES)

真核生物大多数蛋白质合成采用了依赖帽子结构的翻译起始方式.但一组缺乏帽子构的RNA病毒的蛋白质合成起始是依赖其5′端非翻译区（untranslated region,UTR）翻译调控的顺式作用元件——内部核糖体进入位点（internal ribosome entry site, IRES）.它 们能够在一些反式作用因子的辅助下,招募核糖体小亚基到病毒mRNA的翻译起始位点.前,依赖IRES元件翻译起始的RNA病毒在哺乳动物,无脊椎动物及植物中均有发现.因此,对RNA病毒IRES元件的深入研究,不仅有助于阐明相关疾病的发生机理,而且为工业应用和疾病治疗提供借鉴意义.本文对RNA病毒IRES元件发现、分类、结构与功能等作了综述.     

植物蛋白质翻译水平的调控

不同的逆境条件可引起生物机体不同的应答反应,其中PKR(protein kinase double-stranded RNA-dependent)、PERK(PKR-like endoplasmic reticulum kinase)、HRI(heme-regulated inhibitor)和GCN2(general control non-derepressible-2)激活后使真核蛋白翻译起始因子2(e IF2)磷酸化,抑制蛋白质的翻译起始,帮助生物体适应各种逆境条件。雷帕霉素的靶蛋白(TOR)是一个进化上相对保守的丝氨酸/苏氨酸激酶,参与细胞生长与增殖、新陈代谢以及蛋白质的翻译等进程,对细胞的正常生长发育有重要作用。近几年的研究表明,e IF2和TOR介导的信号途径在植物中是保守的,共同参与了蛋白翻译水平的调控。本文综述了植物中e IF2和TOR介导的信号途径对蛋白翻译过程的调控机制,以及蛋白质翻译在植物响应逆境中的重要作用。     

真核翻译起始因子5A在蛋白质合成中的功能及机制

在蛋白质合成过程中,除核糖体、氨酰 tRNA和mRNA外,还有多种翻译因子参与其中。真核翻译起始因子5A（eukaryotic translation initiation factor 5A, eIF5A）是维持细胞活性必不可少的翻译因子,在进化上高度保守。eIF5A是真核细胞中唯一含有羟腐胺赖氨酸（hypusine）的蛋白质,该翻译后修饰对eIF5A的活性至关重要。1978年,人们首次鉴定出eIF5A,认为它在翻译起始阶段促进第1个肽键的形成。直到2013年才证实它主要在翻译延伸阶段调控含多聚脯氨酸基序蛋白质的翻译。在经过四十多年研究后,人们对eIF5A的功能有了新的认识。近期基于核糖体图谱数据的分析表明,eIF5A能够缓解翻译延伸过程中核糖体在多种基序处的停滞,并不局限于多聚脯氨酸基序,并且它还能够通过促进肽链的释放增强翻译终止。此外,eIF5A还可以通过调控某些蛋白质的翻译,间接影响细胞内的各种生命活动。本文综述了eIF5A的多种翻译后修饰、在蛋白质合成和细胞自噬过程中的调控作用以及与人类疾病的关系,并与细菌及古细菌中的同源蛋白质进行了比较,探讨了该因子在进化中的保守性,以期为相关领域的研究提供一定的理论基础。     

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。     

蛋白质修饰与肿瘤糖代谢

蛋白质分子链上接上某种化学基团,从而改变其执行生命复杂的调控和信息传递的功能,这一过程称为"蛋白质修饰".常见的蛋白质翻译后修饰过程有磷酸化、乙酰化、泛素化、糖基化和甲基化等,它们使蛋白质的结构更为复杂,功能更为完善,作用更为专一,调控更为精细.蛋白质修饰在生命体中具有十分重要的作用.在肿瘤代谢中,肿瘤细胞即使在氧气充足的情况下仍进行糖酵解,称为Warburg效应.目前越来越多的研究表明,蛋白质翻译后修饰与肿瘤糖代谢密切相关.本文将就常见的蛋白质修饰方式对肿瘤糖代谢的影响方面的研究进展进行综述.     

Translational control gone awry: a new mechanism of tumorigenesis and novel targets of cancer treatments

Translational control is one of primary regulation mechanisms of gene expression. Eukaryotic translational control mainly occurs at the initiation step, the speed-limiting step, which involves more than ten translation initiation factors [eIFs (eukaryotic initiation factors)]. Changing the level or function of these eIFs results in abnormal translation of specific mRNAs and consequently abnormal growth of cells that leads to human diseases, including cancer. Accumulating evidence from recent studies showed that the expression of many eIFs was associated with malignant transformation, cancer prognosis, as well as gene expression regulation. In the present paper, we perform a critical review of recent advances in understanding the role and mechanism of eIF action in translational control and cancer as well as the possibility of targeting eIFs for therapeutic development.     

Where to begin? The mechanism of translation initiation codon selection in eukaryotes

Selecting the codon at which to begin translation is a complicated event in an already complicated process. Many protein initiation factors (eIFs) have been implicated in start site selection, but the mechanistic details of their activities have remained obscure until recently. Biochemical and genetic studies of eIFs 1, 1A, 2 and 5 have suggested that the 43S pre-initiation complex exists in two conformations and that the changing interactions of the factors within the 43S pre-initiation complex in response to encountering an AUG codon regulates these conformations and, ultimately, the selection of the start codon.     

Small ribosomal protein RPS0 stimulates translation initiation by mediating 40S-binding of eIF3 via its direct contact with the eIF3a/TIF32 subunit

The ribosome translates information encoded by mRNAs into proteins in all living cells. In eukaryotes, its small subunit together with a number of eukaryotic initiation factors (eIFs) is responsible for locating the mRNA's translational start to properly decode the genetic message that it carries. This multistep process requires timely and spatially coordinated placement of eIFs on the ribosomal surface. In our long-standing pursuit to map the 40S-binding site of one of the functionally most complex eIFs, yeast multisubunit eIF3, we identified several interactions that placed its major body to the head, beak and shoulder regions of the solvent-exposed side of the 40S subunit. Among them is the interaction between the N-terminal domain (NTD) of the a/TIF32 subunit of eIF3 and the small ribosomal protein RPS0A, residing near the mRNA exit channel. Previously, we demonstrated that the N-terminal truncation of 200 residues in tif32-Δ8 significantly reduced association of eIF3 and other eIFs with 40S ribosomes in vivo and severely impaired translation reinitiation that eIF3 ensures. Here we show that not the first but the next 200 residues of a/TIF32 specifically interact with RPS0A via its extreme C-terminal tail (CTT). Detailed analysis of the RPS0A conditional depletion mutant revealed a marked drop in the polysome to monosome ratio suggesting that the initiation rates of cells grown under non-permissive conditions were significantly impaired. Indeed, amounts of eIF3 and other eIFs associated with 40S subunits in the pre-initiation complexes in the RPS0A-depleted cells were found reduced; consistently, to the similar extent as in the tif32-Δ8 cells. Similar but less pronounced effects were also observed with the viable CTT-less mutant of RPS0A. Together we conclude that the interaction between the flexible RPS0A-CTT and the residues 200-400 of the a/TIF32-NTD significantly stimulates attachment of eIF3 and its associated eIFs to small ribosomal subunits in vivo.     

The role of IRES trans-acting factors in regulating translation initiation

The majority of mRNAs in eukaryotic cells are translated via a method that is dependent upon the recognition of, and binding to, the methylguanosine cap at the 5' end of the mRNA, by a set of protein factors termed eIFs (eukaryotic initiation factors). However, many of the eIFs involved in this process are modified and become less active under a number of pathophysiological stress conditions, including amino acid starvation, heat shock, hypoxia and apoptosis. During these conditions, the continued synthesis of proteins essential to recovery from stress or maintenance of a cellular programme is mediated via an alternative form of translation initiation termed IRES (internal ribosome entry site)-mediated translation. This relies on the mRNA containing a complex cis-acting structural element in its 5'-UTR (untranslated region) that is able to recruit the ribosome independently of the cap, and is often dependent upon additional factors termed ITAFs (IRES trans-acting factors). A limited number of ITAFs have been identified to date, particularly for cellular IRESs, and it is not yet fully understood how they exert their control and which cellular pathways are involved in their regulation.     

The emerging roles of translation factor eIF4E in the nucleus

The emerging field of nuclear eIF research has yielded many surprises and led to the dissolution of some dogmatic/ideological viewpoints of the place of translation in the regulation of gene expression. Eukaryotic initiation factors (eIFs) are classically defined by their cytoplasmic location and ability to regulate the initiation phase of protein synthesis. For instance, in the cytoplasm, the m7G cap-binding protein eIF4E plays a distinct role in cap-dependent translation initiation. Disruption of eIF4E's regulatory function drastically effects cell growth and may lead to oncogenic transformation. A growing number of studies indicate that many eIFs, including a substantial fraction of eIF4E, are found in the nucleus. Indeed, nuclear eIF4E participates in a variety of important RNA-processing events including the nucleocytoplasmic transport of specific, growth regulatory mRNAs. Although unexpected, it is possible that some eIFs regulate protein synthesis within the nucleus. This review will focus on the novel, nuclear functions of eIF4E and how they contribute to eIF4E's growth-activating and oncogenic properties. Both the cytoplasmic and nuclear functions of eIF4E appear to be dependent on its intrinsic ability to bind to the 5' m7G cap of mRNA. For example, Promyelocytic Leukemia Protein (PML) potentially acts as a negative regulator of nuclear eIF4E function by decreasing eIF4E's affinity for the m7G cap. Therefore, eIF4E protein is flexible enough to utilize a common biochemical activity, such as m7G cap binding, to participate in divergent processes in different cellular compartments.     

eIF3: a versatile scaffold for translation initiation complexes

Translation initiation in eukaryotes depends on many eukaryotic initiation factors (eIFs) that stimulate both recruitment of the initiator tRNA, Met-tRNA(i)(Met), and mRNA to the 40S ribosomal subunit and subsequent scanning of the mRNA for the AUG start codon. The largest of these initiation factors, the eIF3 complex, organizes a web of interactions among several eIFs that assemble on the 40S subunit and participate in the different reactions involved in translation. Structural analysis suggests that eIF3 performs this scaffolding function by binding to the 40S subunit on its solvent-exposed surface rather than on its interface with the 60S subunit, where the decoding sites exist. This location of eIF3 seems ideally suited for its other proposed regulatory functions, including reinitiating translation on polycistronic mRNAs and acting as a receptor for protein kinases that control protein synthesis.     

Protein synthesis in eukaryotic organisms: New insights into the function of translation initiation factor EIF-3

The pathway for initiation of protein synthesis in eukaryotic cells has been defined and refined over the last 25 years using purified components and in vitro reconstituted systems. More recently, powerful genetic analysis in yeast has proved useful in unraveling aspects of translation inherently more difficult to address by strictly biochemical approaches. One area in particular is the functional analysis of multi-subunit protein factors, termed eukaryotic initiation factors (eIFs), that play an essential role in translation initiation. eIF-3, the most structurally complex of the eIFs, has until recently eluded this approach. The identification of the yeast GCD10 gene as the structural gene for the ζ subunit of yeast eIF-3⁽¹⁾ and the analysis of mutant phenotypes has opened the door to the genetic dissection of the eIF-3 protein complex.     

Complex formation between recombinant protein VPg of potato virus Y and heterologous eukaryotic translation initiation factors eIF4E

Genomes of some positive-strand RNA viruses do not contain cap-structure, but instead their 5'-end is covalently linked to a viral protein called VPg. Complex formation between VPg and cellular translation initiation factors (eIFs) has been extensively studied in the context of the model of this complex involvement in virus mRNA translation initiation and cellular protein translation shut down in infected cells. The potato virus (PVY) VPg was expressed in bacterial and baculovirus systems in order to investigate its binding capacity to wheat eIF4E and its isoform. Both purified recombinant eIF4E and eIF(iso)4E were identified in vitro as binding partners of the purified recombinant VPg by using affinity chromatography, as well in vivo by coexpressing of recombinant VPg and eIFs in insect cells with following complex purification using affinity chromatography. Besides it was shown that PVY VPg also formed a complex with endogenous insect eIF4E in vivo. PVY VPg interaction with eIF4E of wheat (non permissive plant for PVY), and also with so evolutionary distant partner as insect eIF4E suggests the conservation of general structural features of eIF4E implicated in the formation of the complex with VPg.     

Elucidating mechanistic principles underpinning eukaryotic translation initiation using quantitative fluorescence methods

Eukaryotic translation initiation is an intricate process involving at least 11 formally classified eIFs (eukaryotic initiation factors), which, together with the ribosome, comprise one of the largest molecular machines in the cell. Studying such huge macromolecular complexes presents many challenges which cannot readily be overcome by traditional molecular and structural methods. Increasingly, novel quantitative techniques are being used to further dissect such complex assembly pathways. One area of methodology involves the labelling of ribosomal subunits and/or eIFs with fluorophores and the use of techniques such as FRET (F?rster resonance energy transfer) and FA (fluorescence anisotropy). The applicability of such techniques in such a complex system has been greatly enhanced by recent methodological developments. In the present mini-review, we introduce these quantitative fluorescence methods and discuss the impact they are beginning to have on the field.     

Curcumin modulates eukaryotic initiation factors in human lung adenocarcinoma epithelial cells

Curcumin, a polyphenolic compound, is the active component of Curcuma longa and has been extensively investigated as an anticancer drug that modulates multiple pathways. Eukaryotic initiation factors (eIFs) have been known to play important roles in translation initiation, which controls cell growth and proliferation. Little is known about the effects of curcumin on eIFs in lung cancer. The objective of this study was to exam the curcumin cytotoxic effect and modulation of two major rate-limiting translation initiation factors, including eIF2α and eIF4E protein expression levels in lung adenocarcinoma epithelial cell line A549. Cytotoxicity was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and protein changes were determined by Western blot. A549 cells were treated with 0–240 μM curcumin for 4–96 h. The inhibitory effects of curcumin on cytotoxicity were dose- and time-dependent (P < 0.001). The 50% inhibitory curcumin concentrations (IC50s) at 24, 48, 72, and 96 h were 93, 65, 40, and 24 μM, respectively. Protein expressions of eIF2α, eIF4E, Phospho-4E-BP1 were down-regulated, while Phospho-eIF2α and Phospho-eIF4E were up-regulated after A549 cells were treated with 20 and 40 μM curcumin for 24 h. In addition, the effects of curcumin on these protein expression changes followed a significant dose-response (P < 0.05, trend test). These findings suggest that curcumin could reduce cell viability through prohibiting the initiation of protein synthesis by modulating eIF2α and eIF4E.