eIF-5A与DHS功能研究及应用进展

脱氧羟腐胺赖氨酸合酶(deoxyhypusine synthase,DHS)在植物、哺乳动物和酵母细胞中普遍存在,参与真核翻译起始因子eIF-5A(eukaryotic initiation factor-5A)的翻译后活化.目前研究表明eIF-5A具有多重生物学功能,如参与细胞增殖、蛋白质翻译、mRNA降解、细胞周期的转化及细胞衰老与凋亡等.eIF-5A是目前已知的DHS的惟一底物,因此研究DHS的功能与作用机理离不开对eIF-5A的功能研究.     

核磁共振波谱法在蛋白质三维结构解析中的应用

蛋白质特定的三维结构与其生物功能密切相关,因此,研究蛋白质的三维结构有助于揭示其生物功能机制。将核磁共振（NMR）波谱法应用于研究溶液状态下蛋白质的三维结构,能够更加准确地揭示蛋白质结构与生物功能之间的关系。本文综述了NMR解析蛋白质三维结构的理论和技术方法,以及NMR结合其他生物物理手段,并辅以分子建模计算法研究蛋白质三维结构的研究进展和最新方法,为精准解析蛋白质的三维结构提供思路及策略。     

蛋白质磁共振——从结构生物学到结构基因组学

在后基因组时代,随着大量物种全基因组序列的获得,结构生物学家面临着结构基因组学的新机遇和挑战。与传统的结构生物学不同的是,结构基因组学的研究主要集中在结构和功能未知并且与从前研究的蛋白质相似性很小的蛋白质。准确的来讲,结构基因组学通过高通量蛋白质表达、结构解析来完成所有蛋白质家族的结构表征,从而能够通过结构预测功能。加州结构基因组学联合实验室发展了高度自动化的蛋白质合成、结晶、结构解析生产线。然而由于一些蛋白质不能被结晶,要想覆盖所有蛋白质结构域还有很大困难。Wuthrich的研究小组通过一些高通量的目的蛋白质筛选和NMR结构解析的方法解决了这一难题。与X射线晶体学解析蛋白质结构相比,NMR技术由于能够解析更接近生理状态的溶液结构而具有互补性。通过获得溶液中的蛋白质稳定性、动力学特征和相互作用信息,正如在朊蛋白和SARS相关蛋白的研究中所表现的那样,NMR技术从扩大已知的蛋白质结构数据库、新的蛋白质功能到化学生物学研究中都扮演着激动人心的角色。     

月季eIF－5A基因的表达增强宿主大肠杆菌对高温和低温的耐性

真核生物翻译起始因子（eIF－5A）是在调控生物生长发育、衰老与环境响应中起重要作用的蛋白质。设计eIF－5A基因的兼并引物,对月季受高温诱导的叶片cDNA进行PCR扩增,获得特异性片段回收、克隆和测序,确定该cDNA为月季eIF-5A(命名为RceIF5A),含有480bp的核苷酸,编码159个氨基酸。将该cDNA序列克隆到原核表达载体PET32a中,获得重组子pET32a－eIF5A。高温（50℃）和低温（4℃）胁迫下含有该基因的大肠杆菌(Escherichia coli) BL21 (pET32a-eIF5A)比E. coli BL21 (pET32a)有明显的抗性提高,据此认为含有重组子的E. coli BL21 (pET32a-eIF5A)对高低温的抗性可能与eIF-5A基因的表达相关。该基因的GeneBank登录号为 EF177192。     

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.     

二维核磁共振谱在多糖结构研究中的应用

二维核磁共振谱（2D NMR）是获取多糖结构信息,尤其是在多糖序列分析方面的有力工具。本文重点介绍了在多糖结构解析中常用的几种2D NMR谱以及2D NMR解析多糖结构的方法。     

鸦胆子的化学成分

对苦木科鸦胆子属植物Brucea javanica（L）Merr的成熟果实鸦胆子提取物的化学成分进行分离鉴定。鸦胆子乙醇提取物经氯仿萃取,硅胶柱色谱分离,通过波谱（^1H NMR,^13C NMR）分析和化学方法鉴定其结构。从中药鸦胆子中分离得到6个苦木内酯类化合物即brusatol（1）、bmeeine D（2）、bruceine E（3）、bruceine F（4）、bmceine A（5）、bruceoside A（6）,另外还有β-谷甾醇（β-sitosterol,7）、胡萝卜苷（daueosterol,8）。     

植物NAC转录因子家族研究概况

植物特有的NAC家族转录因子数量众多, 广泛分布于陆生植物中。NAC转录因子涉及多个生长发育和胁迫应答过程,功能多样而重要, 从发现至今一直是研究的热点。ANAC019蛋白三维结构的解析和一系列NAC基因功能的揭示可以帮助我们更全面地了解NAC家族, 包括它们的起源与分类、生物学功能、表达调控规律以及结构与功能的关系。该文较为详尽地阐述了NAC家族转录因子的研究现状, 并展望其未来的研究方向。     

parp10基因RNA干扰有效靶点的筛选及验证

目的：合成并筛选有效抑制parp10表达的siRNA。方法：根据parp10 cDNA序列,设计并合成prap10基因潜在的RNA干扰（RNAi）片段,将合成的寡核苷酸序列构建到pEGFP-C1H1U6载体中;通过双萤光素酶试验、Western blotting筛选有效的干扰序列;进一步用G418对A549细胞进行耐受度筛选,确定最低耐受度;用G418溶液对转染RNAi重组质粒的A549细胞进行筛选,通过RT-PCR鉴定干扰效果。结果：针对617bp处所构建的RNAi载体能够抑制PARP10的表达,用浓度为400μg/mL G418的McCoy′s 5A培养基筛选转染后的细胞,获得了能够表达绿色荧光标签蛋白的A549细胞株,经RT-PCR检测发现,PARP10表达受到抑制。结论：获得了能够有效抑制PARP10表达的特异性小干扰RNA（siRNA）,为进一步研究其生物学功能提供了条件。     

破解蛋白质翻译机器的结构与功能——2009年诺贝尔化学奖简述

作为将基因信息翻译为蛋白质的机器,核糖体对于生命活动至关重要。2009年诺贝尔化学奖授予了在核糖体结构功能研究中做出突出贡献的三位科学家：Venkatraman Ramakrishnan（英国剑桥MRC分子生物学实验室）,Thomas A. Steitz（耶鲁大学）和Ada E. Yonath（以色列魏茨曼科学研究所）。文章以时间为主线,对核糖体结构以及基于结构的功能研究的历程、意义、历史性突破和应用进行了介绍,并由此对结构生物学的未来进行了一些思考。     

Functional characterization of the Arabidopsis eukaryotic translation initiation factor 5A-2 that plays a crucial role in plant growth and development by regulating cell division, cell growth, and cell death

The eukaryotic translation initiation factor 5A (eIF-5A) is a highly conserved protein found in all eukaryotic organisms. Although originally identified as a translation initiation factor, recent studies in mammalian and yeast (Saccharomyces cerevisiae) cells suggest that eIF-5A is mainly involved in RNA metabolism and trafficking, thereby regulating cell proliferation, cell growth, and programmed cell death. In higher plants, the physiological function of eIF-5A remains largely unknown. Here, we report the identification and characterization of an Arabidopsis (Arabidopsis thaliana) mutant fumonisin B(1)-resistant12 (fbr12). The fbr12 mutant shows an antiapoptotic phenotype and has reduced dark-induced leaf senescence. Moreover, fbr12 displays severe defects in plant growth and development. The fbr12 mutant plant is extreme dwarf with substantially reduced size and number of all adult organs. During reproductive development, fbr12 causes abnormal development of floral organs and defective sporogenesis, leading to the abortion of both female and male germline cells. Microscopic studies revealed that these developmental defects are associated with abnormal cell division and cell growth. Genetic and molecular analyses indicated that FBR12 encodes a putative eIF-5A-2 protein. When expressed in a yeast mutant strain carrying a mutation in the eIF-5A gene, FBR12 cDNA is able to rescue the lethal phenotype of the yeast mutant, indicating that FBR12 is a functional eIF-5A. We propose that FBR12/eIF-5A-2 is fundamental for plant growth and development by regulating cell division, cell growth, and cell death.     

Hypusine is required for a sequence-specific interaction of eukaryotic initiation factor 5A with postsystematic evolution of ligands by exponential enrichment RNA

Hypusine is formed through a spermidine-dependent posttranslational modification of eukaryotic initiation factor 5A (eIF-5A) at a specific lysine residue. The reaction is catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase. eIF-5A is the only protein in eukaryotes and archaebacteria known to contain hypusine. Although both eIF-5A and deoxyhypusine synthase are essential genes for cell survival and proliferation, the precise biological function of eIF-5A is unclear. We have previously proposed that eIF-5A may function as a bimodular protein, capable of interacting with protein and nucleic acid (Liu, Y. P., Nemeroff, M., Yan, Y. P., and Chen, K. Y. (1997) Biol. Signals 6, 166-174). Here we used the method of systematic evolution of ligands by exponential enrichment (SELEX) to identify the sequence specificity of the potential eIF-5A RNA targets. The post-SELEX RNA obtained after 16 rounds of selection exhibited a significant increase in binding affinity for eIF-5A with an apparent dissociation constant of 1 x 10(-7) m. The hypusine residue was found to be critical for this sequence-specific binding. The post-SELEX RNAs shared a high sequence homology characterized by two conserved motifs, UAACCA and AAUGUCACAC. The consensus sequence was determined as AAAUGUCACAC by sequence alignment and binding studies. BLAST analysis indicated that this sequence was present in > 400 human expressed sequence tag sequences. The C terminus of eIF-5A contains a cold shock domain-like structure, similar to that present in cold shock protein A (CspA). However, unlike CspA, the binding of eIF-5A to either the post-SELEX RNA or the 5'-untranslated region of CspA mRNA did not affect the sensitivity of these RNAs to ribonucleases. These data suggest that the physiological significance of eIF-5A-RNA interaction depends on hypusine and the core motif of the target RNA.     

Isolation and characterization of senescence-induced cDNAs encoding deoxyhypusine synthase and eucaryotic translation initiation factor 5A from tomato

Full-length cDNA clones encoding deoxyhypusine synthase (DHS) and eucaryotic initiation factor 5A (eIF-5A) have been isolated from a cDNA expression library prepared from tomato leaves (Lycopersicon esculentum, cv. Match) exposed to environmental stress. DHS mediates the first of two enzymatic reactions that activate eIF-5A by converting a conserved lysine to the unusual amino acid, deoxyhypusine. Recombinant protein obtained by expressing tomato DHS cDNA in Escherichia coli proved capable of carrying out the deoxyhypusine synthase reaction in vitro in the presence of eIF-5A. Of particular interest is the finding that DHS mRNA and eIF-5A mRNA show a parallel increase in abundance in senescing tomato flowers, senescing tomato fruit, and environmentally stressed tomato leaves exhibiting programmed cell death. Western blot analyses indicated that DHS protein also increases at the onset of senescence. It is apparent from previous studies with yeast and mammalian cells that hypusine-modified eIF-5A facilitates the translation of a subset of mRNAs mediating cell division. The present study provides evidence for senescence-induced DHS and eIF-5A in tomato tissues that may facilitate the translation of mRNA species required for programmed cell death.     

A fluorescence study of the interaction of protein synthesis initiation factors 4A, 4E, and 4F with mRNA and oligonucleotide analogs

The initial interaction of mRNA with the protein synthesis machinery presumably involves recognition of the 5'-cap (m7GpppN), although it is not clear at the present time whether this recognition is by eIF-4E or eIF-4F. This process has been studied by direct fluorescence titration experiments. The equilibrium constants for the formation of the binary protein: m7GpppG, protein:mRNA, and protein:protein complexes as well as the ternary mRNA:eIF-4E:eIF-4A complexes were measured. These studies show, for the first time, direct evidence for an eIF-4A:eIF-4E interaction. In contrast to earlier studies, we show that the affinity of eIF-4E and eIF-4F for globin mRNA is similar. Furthermore, the relative affinities of mRNA analogs (capped oligonucleotides) for these initiation factors indicate that the cap is the predominant feature recognized for binding, but other features also contribute to the eIF-4E:mRNA interaction.     

The ATP-dependent interaction of eukaryotic initiation factors with mRNA

The interaction of three protein synthesis initiation factors, eukaryotic initiation factor (eIF)-4A, -4B, and -4F, with mRNA has been examined. Three assays specifically designed to evaluate this interaction are RNA-dependent ATP hydrolysis, retention of mRNAs on nitrocellulose filters, and cross-linking to periodate-oxidized mRNAs. The ATPase activity of eIF-4A is only activated by RNA which is lacking in secondary structure, and the minimal size of an oligonucleotide capable of effecting an optimal activation is 12-18 bases. In the presence of ATP, eIF-4A is capable of binding mRNA. Consistent with the ATPase activity, this binding shows a definite preference for single-stranded RNA. In the absence of ATP, eIF-4F is the only factor to bind capped mRNAs, and this binding, unlike that of eIF-4A, is sensitive to m7GDP inhibition. The activities of both eIF-4A and eIF-4F are stimulated by eIF-4B, which seems to have no specific independent activity in our assays. Evidence from the cross-linking studies indicates that in the absence of ATP, only the 24,000-dalton polypeptide of eIF-4F binds to the 5' cap region of the mRNA. From the data presented in conjunction with the current literature, a suggested sequence of factor binding to mRNA is: eIF-4F is the first initiation factor to bind mRNA ind an ATP-independent fashion; eIF-4B then binds to eIF-4F, if in fact it was not already bound prior to mRNA binding; and finally, eIF-4A binds to the eIF-4F X eIF-4B X mRNA complex and functions in an ATP-dependent manner to allow unwinding of the mRNA.     

The Subcellular Distribution of Eukaryotic Translation Initiation Factor, eIF-5A, in Cultured Cells

To gain insight into the role of the eukaryotic translation initiation factor, eIF-5A, we investigated the subcellular distribution of this protein in several cultured cell types and at different stages of the cell cycle using a highly potent monospecific polyclonal antibody to eIF-5A. Studies using indirect immunofluorescence and confocal microscopy in conjunction with subcellular fractionation demonstrate that eIF-5A is primarily localized in the cytoplasm of cells. This cytoplasmic location of eIF-5A is not significantly altered in different stages of the cell cycle and the subcellular distribution pattern of eIF-5A is not changed by viral oncogene transformation. Cell fractionation experiments identified two populations of eIF-5A in the cytoplasm, a soluble fraction and a fraction bound to internal membranes. By double immunofluorescence staining with an antibody against calnexin, a resident protein of the endoplasmic reticulum (ER), we demonstrate that the membrane-bound fraction of eIF-5A colocalizes with the ER and not with the cytoskeleton. Expression of Rev, a regulatory protein of human immunodeficiency virus type 1 (HIV-1), does not alter the subcellular distribution of endogenous eIF-5A in these cells. eIF-5A is detected in all tissues and cells examined including extracts prepared fromXenopusoocytes. Our results indicate that eIF-5A is a ubiquitous cytoplasmic protein and suggest that a site of eIF-5A function is likely to be in association with the ER.     

Eukaryotic initiation factor 5A is a cellular target of the human immunodeficiency virus type 1 Rev activation domain mediating trans- activation

Expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the presence of the viral trans-activator protein Rev. Rev is localized in the nucleus and binds specifically to the Rev response element (RRE) sequence in viral RNA. Furthermore, the interaction of the Rev activation domain with a cellular cofactor is essential for Rev function in vivo. Using cross-linking experiments and Biospecific Interaction Analysis (BIA) we identify eukaryotic initiation factor 5A (eIF-5A) as a cellular factor binding specifically to the HIV-1 Rev activation domain. Indirect immunofluorescence studies demonstrate that a significant fraction of eIF-5A localizes to the nucleus. We also provide evidence that Rev transactivation is functionally mediated by eIF-5A in Xenopus oocytes. Furthermore, we are able to block Rev function in mammalian cells by antisense inhibition of eIF-5A gene expression. Thus, regulation of HIV-1 gene expression by Rev involves the targeting of RRE-containing RNA to components of the cellular translation initiation complex.     

High-level synthesis in Escherichia coli of functional cap-binding eukaryotic initiation factor eIF-4E and affinity purification using a simplified cap-analog resin

Numerous studies have established the important role that eukaryotic initiation factor-4E (eIF-4E) plays during protein biosynthesis. However, biochemical characterization of eIF-4E has proved difficult, mainly because of its low abundance in cells. To facilitate studies on eIF-4E, we have overexpressed Saccharomyces cerevisiae eIF-4E in Escherichia coli. The isolation of eIF-4E was simplified by using a cap-analog affinity matrix (agarose resin) that is considerably less demanding to prepare than those previously reported. We describe a simple and rapid purification scheme that can yield 2-5 micrograms of a homogenous and active preparation of eIF-4E from 1 ml of E. coli culture. E. coli-expressed eIF-4E is active as determined by its ability to bind the cap structure. The results demonstrate that the cap-binding activity of eIF-4E is not dependent on the presence of other proteins that are present at low levels in eIF-4E preparations isolated from eukaryotic cells.     

Identification of Posttranslationally Modified 18-Kilodalton Protein from Rice as Eukaryotic Translation Initiation Factor 5A

Using anther-derived rice (Oryza sativa L.) cell-suspension cultures, we have identified an 18-kD protein that is posttranslationally modified by spermidine and is influenced by endogenous polyamine levels. The posttranslationally modified residue has been identified as the unusual amino acid hypusine [N[epsilon]-(4-amino-2-hydroxybutyl)lysine] by reverse-phase high-performance liquid chromatography and gas chromatography-mass-spectrometry analyses. Differential labeling of the protein with labeled amines provided evidence that the butylamine moiety of spermidine is the immediate precursor of the hypusine residue in the protein. The eukaryotic translation initiation factor 5A (eIF-5A) is the only known mammalian protein that undergoes a similar posttranslational modification with hypusine. The purified 18-kD protein co-electrophoreses with human translational initiation factor eIF-5A in both isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels. The purified protein from rice stimulated methionyl-puromycin synthesis in vitro, indicating its functional similarity to mammalian eIF-5A. The results presented provide evidence that the posttranslationally modified 18-kD protein from rice containing hypusine is eIF-5A and suggest the conservation of hypusine-containing translation initiation factor eIF-5A in eukaryotes.