Structure comparison and evolutionary relations between elongation factors EF-Tu (EF-1α) and SUP 2 proteins

On the basis of high homology and structural similarity, three genes, SUP2 Saccharomyces cerevisiae, SUP2 Pichia pinus and GST1 Homo sapiens, might be considered as members of one family named SUP2. Comparison of the primary structure of SUP2 proteins and elongation factors EF-Tu(EF-1) from 19 different species was performed. It was found that SUP2 proteins bear more homology to eukaryotic elongation factor than to procaryotic EF-Tu, though the degree of sequence conservation in SUP2 proteins is smaller than in EF-1 factors. The extensive phylogenetic analysis of SUP2 and EF-Tu(EF-1) genes was performed by means of 3 methods, 2 phenetic and one cladystic (maximal parsimony). The data support the close relation of SUP2 genes to other elongation factor genes.     

奥利亚罗非鱼EF-1α基因的克隆与结构分析

真核生物延伸生长因子基因(EF-1α)在蛋白质翻译过程中起着重要的作用,其序列具有高度的保守性,是一种管家基因.本文通过 RT-PCR 克隆出奥利亚罗非鱼(Oreochromis aureus)EF-1α的部分cDNA序列,其长度为425 bp,翻译成141个氨基酸,计算的蛋白质分子量为15.1 ku.同源性分析显示,奥利亚罗非鱼EF-1α氨基酸序列与尼罗罗非鱼(O.niloticus)的相似性最高,为100%;与青鳉(Oryzias latipes)、欧洲鲈(Dicentrarchus labrax)、斑马鱼(Danio rerio)、鲑鱼(Salmo trutta)的相似性分别为92%、91%、85%、82%;与小鼠(Mus musculus)、大鼠(Rattus norvegicus)、人(Homo sapiens)、鸡(Gallus gallus)的相似性均为85%.同时克隆出奥利亚罗非鱼EF-1α相应的DNA序列,共506 bp.cDNA与DNA的序列比对显示克隆出的奥利亚罗非鱼EF-1α含有1个内含子,这为将来设计EF-1α荧光定量引物以及测定其在不同组织中的表达量变化打下基础.     

Exchange of free GTP with EF-1α·GDP complex promoted by a factor EF-1β from pig liver

Eukaryotic polypeptide chain elongation factor 1 (EF-1) has been resolved into two complementary factors, EF-1α and EF-1β, both of which were purified. Recently, we find that [³H] GDP bound to purified EF-1α is replaced by exogenous GTP rather slowly when the reaction is carried out at ionic strength optimal for polyphenylalanine synthesis. EF-1β stimulates the exchange of free GTP with EF-1α·GDP, indicating that the function of EF-1β is, at least in part, similar to that of bacterial EF-Ts.     

Multiple Phosphorylation Sites and Quaternary Organization of Guanine-Nucleotide Exchange Complex of Elongation Factor-1 (EF-1βγδ/ValRS) Control the Various Functions of EF-1α

The eukaryotic guanine-nucleotide exchange factor commonly called elongation factor-1 (EF-1), comprises four different subunits including valyl-tRNA synthetase (EF-1/ValRS). The factor is multiply-phosphorylated by three different protein kinases, protein kinase C, casein kinase II and cyclin dependent kinase 1 (CDK1). EF-1/ValRS is organized as a macromolecular complex for which we propose a new structural model. Evidence that EF-1/ValRS is a sophisticated supramolecular complex containing many phosphorylation sites, makes it a potential regulator of any of the functions of its partner EF-1, not only involved in protein synthesis elongation, but also in many other cellular functions.     

α1- and α5-containing Laminins Regulate the Development of Bile Ducts via β1 Integrin Signals

Signals derived from basal lamina components are important for developing three-dimensional architecture of epithelial tissues. Laminins consisting of α, β, and γ subunits in basal lamina play pivotal roles in the formation and maintenance of epithelial tissue structures. However, it remains unclear which laminin isoforms transmit signals and how epithelial cells receive them to regulate multiple developmental processes. In three-dimensional culture of a liver progenitor cell line, Hepatic Progenitor Cells Proliferating on Laminin (HPPL), the cells establish apicobasal polarity and form cysts with a central lumen. Neutralizing antibody against β1 integrin blocked the formation and maintenance of the cyst structure, indicating that β1 integrin signaling was necessary throughout the morphogenesis. Although the addition of α1-containing laminin, a ligand of β1 integrin, induced cyst formation, it was dispensable for the maintenance of the cyst, suggesting that HPPL produces another ligand for β1 integrin to maintain the structure. Indeed, we found that HPPL produced α5-containing laminin, and siRNA against laminin α5 partially inhibited the lumen formation. In fetal liver, p75NTR(+) periportal fibroblasts and bile duct epithelial cells, known as cholangiocytes, expressed α1- and α5-containing laminins, respectively. In laminin α5 KO liver, cholangiocytes normally emerged, but the number of bile ducts was decreased. These results suggest that α1-containing laminin is sufficient as a component of the basal lamina for the commitment of bipotential liver progenitors to cholangiocytes and the apicobasal polarization, whereas α5-containing laminin is necessary for the formation of mature duct structures. Thus, α1- and α5-containing laminins differentially regulate the sequential events to form epithelial tissues via β1 integrin signals.     

真核表达的EF-1α-Flag蛋白对体外蛋白翻译的影响

目的:构建EF-1α-Flag到pcDNA3.0表达载体上,在哺乳动物细胞中表达并纯化延伸因子1α(EF-1α),测定其对体外蛋白翻译的影响。方法:通过PCR技术从293T细胞cDNA文库中扩增EF-1α基因片段,连接到pcDNA3.0载体上,经电泳、测序和转入细胞中检测EF-1α-Flag蛋白表达等方式验证所构建的重组质粒是否正确,然后经Flag肽置换法纯化出EF-1α蛋白用于体外蛋白翻译实验,萤光素酶活性检测翻译出的蛋白含量。结果:测序和蛋白表达鉴定结果表明扩增的EF-1α-Flag基因序列正确,所构建的重组载体在哺乳动物细胞中能获得表达;考马斯亮蓝染色发现经Flag肽置换的EF-1α蛋白纯度较高,在体外蛋白翻译实验中EF-1α蛋白能促进蛋白的翻译。结论:从哺乳动物细胞中纯化的EF-1α蛋白能促进蛋白翻译,可能与其作为翻译延伸因子相关,为进一步研究EF-1α的功能奠定了基础。     

Complex distribution of EFL and EF-1α proteins in the green algal lineage

Background  

EFL (or elongation factor-like) is a member of the translation superfamily of GTPase proteins. It is restricted to eukaryotes, where it is found in a punctate distribution that is almost mutually exclusive with elongation factor-1 alpha (EF-1α). EF-1α is a core translation factor previously thought to be essential in eukaryotes, so its relationship to EFL has prompted the suggestion that EFL has spread by horizontal or lateral gene transfer (HGT or LGT) and replaced EF-1α multiple times. Among green algae, trebouxiophyceans and chlorophyceans have EFL, but the ulvophycean Acetabularia and the sister group to green algae, land plants, have EF-1α. This distribution singles out green algae as a particularly promising group to understand the origin of EFL and the effects of its presence on EF-1α.     

Novel peptide inhibiting both TEM-1 β-lactamase and penicillin-binding proteins

9G4H9, a catalytic antibody displaying β-lactamase-like activity, has been developed by the anti-idiotypic approach using β-lactamase as the first antigen. Thus 9G4H9 represents the 'internal image' of β-lactamase. We selected a cyclic peptide anchored to a bacteriophage M13 library using 9G4H9 as the target. Pep90 is a cyclic heptapeptide enclosed between two cysteine residues. We showed that Pep90 could inhibit both TEM-1 β-lactamase (K(i) = 333 μm) and several penicillin-binding proteins (IC?? values ranging from 6-62 μm). We determined that the tryptophan residue of Pep90 is of crucial importance for its inhibitory activity. Using Pep90 as a scaffold, we generated a new class of peptidomimetics that retained inhibitory activity towards TEM-1 β-lactamase.     

基于COⅡ和EF-1α基因部分序列的中国蝶类科间系统发生关系

为了探讨中国蝶类科间系统发生关系,本文对细胞色素氧化酶Ⅱ(COⅡ)的部分序列(620bp)和延伸因子基因(EF-1α)部分序列(595bp)进行了分析(共1215bp)。其中有464个变异位点,330个简约信息位点。COⅡ基因部分序列表现出明显的A T含量(76.3%)偏高。分别采用最大简约法(Maximum Parsimony,MP)、最大似然法(Maximum Likelihood,ML)和贝叶斯推论法(Bayesian Inference,BI)重建了分子系统树。结果表明:弄蝶科、凤蝶科、粉蝶科、灰蝶科能够单独成一支,其中弄蝶科位于系统树的基部,分化较早,是较原始的类群,与传统的形态分类结果是相一致的;粉蝶科与凤蝶科的亲缘关系较近;蚬蝶科倾向与灰蝶科有较近的亲缘关系,且蚬蝶科种群始终聚为一支,显示了该科是一个单系群,从单系性方面来看,本文支持将蚬蝶科作为一个独立的科;此外,本文结果表明,中国分布的蛱蝶总科是一单系群,并且它与灰蝶科和蚬蝶科聚成的一支是姐妹群关系。     

基于COI和EF-1α基因的绢粉蝶属和妹粉蝶属系统发育关系分析

【目的】由于绢粉蝶属Aporia和妹粉蝶属Mesapia的分类地位尚存在争议,本研究基于COI和EF-1α基因探讨它们的系统发育关系。【方法】对采自中国的19个种(含绢粉蝶属13个种,妹粉蝶属1种以及用作外群的另外3个属的5个种)的COI和EF-1α基因部分序列进行了测定和分析;依据这2个基因的联合序列采用最大似然法(maximum likelihood,ML)和贝叶斯法(Bayesian inference,BI)构建了这些种的系统发育树。【结果】序列分析结果显示,测得的COI序列长度为657 bp,EF-1α序列长度为642 bp,联合后获得的序列总长为1 299 bp,其中变异位点439个,简约信息位点249个;序列A+T的含量明显高于C+G的含量。系统发生分析结果显示,除外群外,其余种类形成单系群(BV=100,PP=1.00),且分成两大支,一支为(奥倍绢粉蝶A.oberthuri+锯纹绢粉A.goutellei)+(丫纹绢粉蝶A.delavayi+(完善绢粉蝶A.agathon+(马丁绢粉蝶A.martineti+(大翅绢粉蝶A.largeteaui+巨翅绢粉蝶A.gigantea)))),另一支为普通绢粉蝶A.genestieri+(中亚绢粉蝶A.leucodice+((绢粉蝶A.crataegi+灰翅绢粉蝶A.potanini)+(妹粉蝶M.peloria+(暗色绢粉蝶A.bieti+小檗绢粉蝶A.hippia))))。【结论】本研究结果支持妹粉蝶属应为绢粉蝶属的异名,且绢粉蝶属内不再划分亚属和种组。     

The molecular systematics of blowflies and screwworm flies (Diptera: Calliphoridae) using 28S rRNA, COX1 and EF-1α: insights into the evolution of dipteran parasitism

The Calliphoridae include some of the most economically significant myiasis-causing flies in the world - blowflies and screwworm flies - with many being notorious for their parasitism of livestock. However, despite more than 50 years of research, key taxonomic relationships within the family remain unresolved. This study utilizes nucleotide sequence data from the protein-coding genes COX1 (mitochondrial) and EF1α (nuclear), and the 28S rRNA (nuclear) gene, from 57 blowfly taxa to improve resolution of key evolutionary relationships within the family Calliphoridae. Bayesian phylogenetic inference was carried out for each single-gene data set, demonstrating significant topological difference between the three gene trees. Nevertheless, all gene trees supported a Calliphorinae-Luciliinae subfamily sister-lineage, with respect to Chrysomyinae. In addition, this study also elucidates the taxonomic and evolutionary status of several less well-studied groups, including the genus Bengalia (either within Calliphoridae or as a separate sister-family), genus Onesia (as a sister-genera to, or sub-genera within, Calliphora), genus Dyscritomyia and Lucilia bufonivora, a specialised parasite of frogs and toads. The occurrence of cross-species hybridisation within Calliphoridae is also further explored, focusing on the two economically significant species Lucilia cuprina and Lucilia sericata. In summary, this study represents the most comprehensive molecular phylogenetic analysis of family Calliphoridae undertaken to date.     

Cholecystokinin-Mediated RhoGDI Phosphorylation via PKCα Promotes both RhoA and Rac1 Signaling

RhoA and Rac1 have been implicated in the mechanism of CCK-induced amylase secretion from pancreatic acini. In all cell types studied to date, inactive Rho GTPases are present in the cytosol bound to the guanine nucleotide dissociation inhibitor RhoGDI. Here, we identified the switch mechanism regulating RhoGDI1-Rho GTPase dissociation and RhoA translocation upon CCK stimulation in pancreatic acini. We found that both Gα13 and PKC, independently, regulate CCK-induced RhoA translocation and that the PKC isoform involved is PKCα. Both RhoGDI1 and RhoGDI3, but not RhoGDI2, are expressed in pancreatic acini. Cytosolic RhoA and Rac1 are associated with RhoGDI1, and CCK-stimulated PKCα activation releases the complex. Overexpression of RhoGDI1, by binding RhoA, inhibits its activation, and thereby, CCK-induced apical amylase secretion. RhoA translocation is also inhibited by RhoGDI1. Inactive Rac1 influences CCK-induced RhoA activation by preventing RhoGDI1 from binding RhoA. By mutational analysis we found that CCK-induced PKCα phosphorylation on RhoGDI1 at Ser96 releases RhoA and Rac1 from RhoGDI1 to facilitate Rho GTPases signaling.     

The HIF-1α-c-Myc pathway and tumorigenesis

Comment on: Hayashi M, et al. Cell Cycle 2011; 10:2364–72     

Antagonistic crosstalk between APC and HIF-1α

Most colorectal cancers have mutations in the tumor suppressor APC. The best-understood function of APC is its participation in a protein complex that regulates the availability of β-catenin. Solid tumors are characterized by the presence of hypoxia as well as inflammation, which leads to the upregulation of Hypoxia Inducible Factors like HIF-1α. We recently demonstrated a novel antagonistic link between APC and HIF-1α. We found that hypoxia results in reduced levels of APC mRNA and protein via a direct HIF-1α-dependent mechanism. Similarly, APC mediates the repression of HIF-1α. However, this requires wild-type APC, low levels of β-catenin and NFκB activity. These results reveal the downregulation of APC as a novel mechanism that contributes to the survival advantage induced by hypoxia and cytokines such as TNFα. Our data indicate that loss-of-function mutations in APC result in the engagement of the hypoxia response. Importantly, this suggests that other stimuli that induce HIF, such as inflammatory cytokines and oncogenes, alter APC function.