真核生物起始因子5

真核生物起始因子5(eIF-5)是一种重要的翻译起始因子,过去人们认为它只是GTP酶活化因子,催化eIF-2上的GTP水解,促进80 S起始复合体的形成.近年来人们发现它不仅可以催化eIF-2上的GTP水解,还参与eIF-3功能的发挥,与eIF-2、eIF-3同时结合,促进起始因子复合体的形成.     

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。     

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.     

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。     

Isolation of Eukaryotic Initiation Factor 2 from Rat Brain

Eukaryotic initiation factor 2 (eIF-2) was isolated from salt-washed microsomes of 4-day-old rat brain which show a high rate of protein synthesis. A three-step purification scheme was employed, including heparin-Sepharose, phosphocellulose, and DEAE-cellulose column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated factor revealed three polypeptides with molecular weights of 43,000, 54,000, and 59,000 and 90% purity. The rat brain eIF-2 forms ternary complexes with [3H]methionyl-tRNAi and GTP. In terms of specific activity, the purification does not correspond to that revealed by electrophoretic analysis. During purification there is an apparent loss of additional factors that modulates the activity of eIF-2 and explains the high rate of activity of the crude fraction.     

Eukaryotic expression and secretion of EGFP-labeled annexin A5

The Ca²⁺-dependent binding of annexin A5 to phosphatidylserine on cell surfaces is a reliable marker for apoptosis that is widely used in flow cytometry based apoptosis assays. In this approach, annexin A5 must be coupled to a fluorescent dye, but standard dyes such as fluorescein are photolabile, and the heterogeneous chemical linkage partially inhibits binding to phosphatidylserine. Recombinant fusions comprising annexin A5 and fluorescent proteins are available for prokaryotic expression, but can be purified only at low concentrations due to their low solubility in the cytoplasm. Here we describe a eukaryotic expression system for the secretion of functional recombinant annexin A5, with and without fluorescent protein fusions, in different formats. Metal affinity purification yielded up to 18 μg of histidine-tagged annexin A5 fusions per ml processed cell culture supernatants. Furthermore the supernatant itself was sufficient for direct use in apoptosis assays. The availability of such fusion proteins offers new and more economical opportunities for the development and application of this widely utilized apoptosis assay.     

Human Eukaryotic Initiation Factor 2 (eIF2)-GTP-Met-tRNAi Ternary Complex and eIF3 Stabilize the 43 S Preinitiation Complex

The formation of a stable 43 S preinitiation complex (PIC) must occur to enable successful mRNA recruitment. However, the contributions of eIF1, eIF1A, eIF3, and the eIF2-GTP-Met-tRNA_i ternary complex (TC) in stabilizing the 43 S PIC are poorly defined. We have reconstituted the human 43 S PIC and used fluorescence anisotropy to systematically measure the affinity of eIF1, eIF1A, and eIF3j in the presence of different combinations of 43 S PIC components. Our data reveal a complicated network of interactions that result in high affinity binding of all 43 S PIC components with the 40 S subunit. Human eIF1 and eIF1A bind cooperatively to the 40 S subunit, revealing an evolutionarily conserved interaction. Negative cooperativity is observed between the binding of eIF3j and the binding of eIF1, eIF1A, and TC with the 40 S subunit. To overcome this, eIF3 dramatically increases the affinity of eIF1 and eIF3j for the 40 S subunit. Recruitment of TC also increases the affinity of eIF1 for the 40 S subunit, but this interaction has an important indirect role in increasing the affinity of eIF1A for the 40 S subunit. Together, our data provide a more complete thermodynamic framework of the human 43 S PIC and reveal important interactions between its components to maintain its stability.     

Eukaryotic elongation factor 2 kinase confers tolerance to stress conditions in cancer cells

Eukaryotic elongation factor 2 (eEF2) is a member of the GTP-binding translation elongation factor family that is essential for protein synthesis. eEF2 kinase (eEF2K) is a structurally and functionally unique protein kinase in the calmodulin-mediated signaling pathway. eEF2K phosphorylates eEF2, thereby inhibiting eEF2 function under stressful conditions. eEF2K regulates numerous processes, such as protein synthesis, cell cycle progression, and induction of autophagy and apoptosis in cancer cells. This review will demonstrate the mechanisms underlying eEF2K activity in cancer cells under different stresses, such as nutrient deprivation, hypoxia, and DNA damage via eEF2 regulation. In vivo, in vitro, and clinical studies indicated that eEF2K may be a novel biomarker and therapeutic target for cancer.     

Molecular Cloning and Characterization of a Gene Encoding Eukaryotic Initiation Factor iso4E in Tomato (Solanum lycopersicum)

The eukaryotic translation initiation factor 4E (eIF4E) and its isoform, eIF(iso)4E, play important roles in protein translation and recently reported to be involved in plant–virus interactions. A cDNA encoding the tomato eIF(iso)4E was cloned based on a tentative consensus (TC170275) in TIGR (), and was designated as SleIF(iso)4E, with an open reading frame of 603 nucleotides encoding a protein of 200 amino acids. The calculated molecular weight of the SleIF(iso)4E protein was 22.85 kD, and the theoretical isoelectric point was 5.76. The amino acid sequence of SleIF(iso)4E showed 66–91% identity with eIF(iso)4Es in pepper, tobacco, pea and maize, and 44–51% identity with eIF4Es from other plants. The phylogenetic relationship and tertiary structure comparisons indicate that SleIF(iso)4E share high homology and strict conserved regions with other members of the eIF4E family, a characteristic of all members of this family. Semi-quantitative RT-PCR showed varying expression levels of SleIF(iso)4E in different tissues. By comparing eIF(iso)4E coding sequences between resistant and susceptible tomato genotypes, correlation between sequence variations and virus resistance was identified. These findings provide good grounds for future research on the role of SleIF(iso)4E in translation initiation and plant–virus interactions. Sequence data of SleIF(iso)4E from this article have been deposited at GenBank under accession number EU119958.     

Effects of Essential Amino Acid Deficiency on General Control Nonderepressible 2/Eukaryotic Initiation Factor 2 Signaling and Proteomic Changes in Primary Bovine Mammary Epithelial Cells

We hypothesized that the general control nonderepressible 2 (GCN2)/eukaryotic initiation factor 2 (eIF2) signaling pathway and intracellular protein synthesis (PS) are regulated to maintain milk PS in primary bovine mammary epithelial cells (MECs) under essential amino acid (EAA) starvation conditions. We cultured MECs with 0%, 2% (depletion), and 100% (control) EAA for two exposure times (8 and 24 h), followed by three refeeding (RF) times with 100% EAA (0, 8, and 24 h). Subsequently, we measured cell viability, total protein concentration, and proliferation. Western blotting was used to quantify the levels of casein and the expression of total GCN2 and eIF2, as well as phosphorylated GCN2 (GCN2P) and eIF2 (eIF2P). The ISOQuant method was used to assess MEC proteomes, and the resultant data were analyzed using the Kruskal–Wallis test, nonpaired Wilcoxon rank post-hoc test, and ANOVA–Tukey test, as well as principal component analyses and multiple regressions models. Differences in cell viability were observed between the control versus the depleted and repleted MECs, respectively, where 97.2–99.8% viability indicated low cell death rates. Proliferation (range, 1.02–1.55 arbitrary units (AU)) was affected by starvation for 12 and 24 h and repletion for 24 h, but it was not increased compared with the control. Total protein expression was unaffected by both depletion and repletion treatments (median 3158 µg/mL). eIF2P expression was significantly increased (p < 0.05) after treatment with 2% EAA for 8 and 24 h compared with 2% EAA with 8 h + 24 h RF and 2% EAA with 24 h + 8 h RF. GCN2P also showed significantly increased expression (p < 0.05) after treatment with 2% EAA for 24 h compared with the control and 2% EAA with 24 h + 8 h RF. Intracellular casein/α-tubulin expression was unaffected by 2% EAA compared with control (0.073 ± 0.01 AU versus 0.086 ± 0.02 AU, respectively). We studied 30 of the detected 1180 proteins, 16 of which were differentially expressed in starved and refed MECs. Cells faced with EAA deficiency activated the GCN2P/eIF2P pathway, and the lack of change in the levels of casein and other milk proteins suggested that the EAA deficit was mitigated by metabolic flexibility to maintain homeostasis.     

转录因子AP-2及其在癌症中的作用

本文综述了关于AP-2基因家族的最近期研究进展。AP-2是一个分子量为52-kDa的转录因子,有其独特的蛋白质分子结构,其N端是富含脯氨酸和谷氨酸的转录激活域,C端的helix-span-helix结构可以形成二聚体并与特异的DNA序列结合。AP-2家族都能以同源或异源二聚体的方式与一段保守序列5’-GCCNNNGGC-3’结合。AP-2α是最早被鉴定和研究的家族成员,很多研究表明AP-2α是一个抑癌基因。     

小麦蛋白翻译起始因子5A基因（eIF5A）的克隆与分析

真核生物的翻译起始因子5A （eIF5A）是调控生物生长发育、衰老及环境适应等的重要因子。利用设计的小麦蛋白翻译起始因子5A基因的引物对小麦“中国春”基因组DNA和cDNA进行PCR扩增,并将扩增的特异片段回收、克隆和测序,从基因组DNA中得到长度分别为1 679 bp、1 910 bp两条带,从cDNA扩增得到1条636 bp带,分别命名为eIF5a1（基因登录号：DQ167202）、eIF5a2（基因登录号：DQ167201）和eIF5a3。利用GeneRace方法得到eIF5a3（基因登录号：DQ167203）的全长为768 bp。序列分析表明,eIF5a1、eIF5a2具82.3%相似性,都形成636 bp的转录产物,转录产物仅6个核苷酸差异。将eIF5a1、eIF5a2和 eIF5a3这3个序列的预测氨基酸序列进行比对,发现仅有1～2个氨基酸的差异,证实它们为eIF5A基因家族的成员。进化分析表明它们与报道的玉米、水稻、西红柿、烟草的eIF5A基因序列的遗传关系最近。进一步研究表明eIF5a2位于2B染色体上,并用半定量RT-PCR 研究了小麦eIF5A基因的表达情况。     

柽柳翻译起始因子(eIF-5A)基因的克隆及原核表达

根据柽柳cDNA文库中获得的eIF-5A基因片段,用RACE技术克隆出其全长cDNA序列.cDNA长度为799 bp,编码159个氨基酸.将该cDNA序列克隆到原核表达载体pET28a中,获得重组质粒pET28a-eIF5A.不同浓度NaCl胁迫下大肠杆菌(Escherichia coli)BL21(pET28a-eIF5A)比E.coli BL21(pET28a)有明显的抗盐性,前者菌株存活率在1.0 mo1·L-1NaCl盐胁迫下是后者的9.3倍,据此认为E.coliBL21(pET28a-eIF5A)的耐盐性可能与eIF-5A基因的表达相关.该基因的GenBank登录号为AY587771(基因)、AAT01416(蛋白).     

Increased Activity of Eukaryotic Initiation Factor 2B in PC12 Cells in Response to Differentiation by Nerve Growth Factor

Abstract: Translational rates, and activities and levels of initiation factors 2 and 2B were assessed in rat pheochromocytoma cells upon nerve growth factor (NGF) treatment. Two or 5 days of exposure to NGF caused significant quantitative increases in protein synthesis rate that are deemed necessary for neuronal differentiation. Changes in initiation factor 2 activity, as measured by its capacity to form a ternary complex, occur parallel to the observed changes in protein synthesis. Nevertheless, neither the intracellular levels of the initiation factor 2 nor the degree of phosphorylation of its α subunit can justify this increased activity. Interestingly, initiation factor 2B activity increases parallel to the neurite outgrowth, being significantly higher after 5 days of exposure to NGF, and could be responsible for the elevated rate of protein synthesis. No significant changes in the levels of eukaryotic initiation factor 2B, as determined with two different antibodies against the γ and ε subunits of the factor, were observed, implying that the increased activity should be regulated by factors other than its cellular concentration. Our results support the hypothesis that initiation factor 2B may play a role in the biochemical events controlling the differentiative growth factor-induced signaling pathway in these cells.