The hepatitis C virus E1 glycoprotein undergoes productive folding but accelerated degradation when expressed as an individual subunit in CHO cells

Hepatitis C Virus E1E2 heterodimers are components of the viral spike. Although there is a general agreement on the necessity of the co-expression of both E1 and E2 on a single coding unit for their productive folding and assembly, in a previous study using an in vitro system we obtained strong indications that E1 can achieve folding in absence of E2. Here, we have studied the folding pathway of unescorted E1 from stably expressing CHO cells, compared to the folding observed in presence of the E2 protein. A DTT-resistant conformation is achieved by E1 in both situations, consistent with the presence of an E2-independent oxidative pathway. However, while the E1E2 heterodimer is stable inside cells, E1 expressed alone is degraded within a few hours. On the other hand, the oxidation and stability of individually expressed E2 subunits is dependent on E1 co-expression. These data are consistent with E1 and E2 assisting each other for correct folding via different mechanisms: E2 assists E1 by stabilizing a semi-native conformation meanwhile E1 drives E2 towards a productive folding pathway.     

Pyruvate dehydrogenase kinase activity of pig heart pyruvate dehydrogenase (E1 component of pyruvate dehydrogenase complex).

The pyruvate dehydrogenase (E1) and acetyltransferase (E2) components of pig heart and ox kidney pyruvate dehydrogenase (PDH) complex were separated and purified. The E1 component was phosphorylated (alpha-chain) and inactivated by MgATP. Phosphorylation was mainly confined to site 1. Addition of E2 accelerated phosphorylation of all three sites in E1 alpha and inactivation of E1. On the basis of histone H1 phosphorylation, E2 is presumed to contain PDH kinase, which was removed (greater than 98%) by treatment with p-hydroxymercuriphenylsulphonate. Stimulation of ATP-dependent inactivation of E1 by E2 was independent of histone H1 kinase activity of E2. The effect of E2 is attributed to conformational change(s) induced in E1 and/or E1-associated PDH kinase. PDH kinase activity associated with E1 could not be separated from it be gel filtration or DEAE-cellulose chromatography. Subunits of PDH kinase were not detected on sodium dodecyl sulphate/polyacrylamide gels of E1 or E2, presumably because of low concentration. The activity of pig heart PDH complex was increased by E2, but not by E1, indicating that E2 is rate-limiting in the holocomplex reaction. ATP-dependent inactivation of PDH complex was accelerated by E1 or by phosphorylated E1 plus associated PDH kinase, but not by E2 plus presumed PDH kinase. It is suggested that a substantial proportion of PDH kinase may accompany E1 when PDH complex is dissociated into its component enzymes. The possibility that E1 may possess intrinsic PDH kinase activity is considered unlikely, but may not have been fully excluded.     

五种大戟属植物nrDNA的ITS序列分析及其叶的比较解剖学研究

运用PCR直接测序法和石蜡切片法,测定5种安徽产大戟属植物的nrDNA的内转录间隔区(ITS)序列包括5.8SrDNA及其叶片的解剖。结果表明(1)5种大戟属植物的ITS的长度范围为641~662bp,运用Maga软件构件ITS树把5种植物归为两大支大戟、月腺大戟与乳浆大戟聚为一支;地锦和斑地锦聚为另一支。结果表明地锦和斑地锦亲缘关系较近,而大戟、月腺大戟和乳浆大戟亲缘关系较近。(2)5种大戟属植物的叶片结构中,除地锦和斑地锦外,其余3种有明显的栅栏组织和海绵组织之分,但二者在叶肉中的厚度比在种间有一定的差别;而地锦和斑地锦叶的栅栏组织与海绵组织分化不明显,且皆有乳汁管,其他3种植物叶片内未见此结构,叶片结构分析表明大戟、月腺大戟和乳浆大戟叶片结构相近,而地锦草和斑地锦叶片结构也相近。(3)分析表明nrDNA的ITS序列及叶的比较解剖特征具有明显的相关性。以上研究结果为大戟属植物的分类和药用开发提供了实验证据。     

Heterogeneity of glyceraldehyde-3-phosphate dehydrogenase from human brain

In an attempt to characterize enzymes from human brain capable of dehydrogenating short chain aliphatic aldehydes, four groups of enzymes which catalyze inorganic phosphate-dependent reversible dehydrogenation of glyceraldehyde 3-phosphate as well as short chain aldehydes have been purified and characterized. Three enzyme groups are visualized as multiple bands on isoelectric focusing: E6.6 (pI 6.65, 6.75, 6.85); E6.8 (pI 6.8, 6.9); E8.5 (pI 8.5, 8.6); one enzyme, E9.0, is seen as a single band pI 9.0. The subcellular localization of E6.8, E8.5 and E9.0 appears to be mitochondrial. The mitochondrial enzymes differ slightly in molecular weight: E6.8 is 142,000 with subunits of 36,000 and 38,000; E8.5 is 120,000 with a subunit weight of 29,500; E9.0 is 133,000 with a subunit of 33,000. The E8.5 and E9.0 enzymes also appear to contain Zr as part of their molecular structure. E6.6 (subcellular localization uncertain) is a dimer with a molecular weight of 98,000 and two subunits of 58,000 and 61,000. The specific activity with glyceraldehyde-3-phosphate is: E6.6, 8.6 IU/mg; E6.8, 13 IU/mg; E8.5, 158 IU/mg; E9.0, 620 IU/mg. With glyceraldehyde 3-phosphate and 1,3-diphosphoglyceric acid and Km values of all the enzymes are similar (10-40 microM), except for E6.8 whose Km for glyceraldehyde 3-phosphate is very sensitive to pH and is extremely low at pH 7.0 (2 microM), while being considerably higher than that for the other enzymes at pH 9.0 (170 microM). The molecular properties, Km values as well as high specific activity with glyceraldehyde 3-phosphate identify E6.8, E8.5 and E9.0 as glyceraldehyde-3-phosphate dehydrogenases (EC 1.2.1.12). The catalytic properties of E6.6 are similar to those of E6.8, E8.5 and E9.0, but its molecular properties are different, precluding definite identification.     

New species of Elaphoglossum (Elaphoglossaceae) from northern South America

The fern genusElaphoglossum is well-represented in the Venezuelan pteridoflora with 98 species. Careful observation of the indument of rhizome and blade is necessary to distinguish the taxa. Thirty-three species and one variety are here describe as new:E. anceps, E. appressum, E. atrorubens, E. atrosquamatum, E. chrysopogon, E. crispatum var.crispatum, E. crispatum var.beitelii, E. delicatulum, E. dolichopus, E. drewianum, E. eriopus, E. floccosum, E. grallator, E. hieracioides, E. incubus, E. luteynii, E. maguirei, E. nigrocostatum, E. obovatum, E. ornithoglossum, E. ortegae, E. pilosius, E. praetermissum, E. stenoglossum, E. stergiossi, E. steyermarkii, E. styriacum, E. succubus, E. tachirense, E. tantalinum, E. urophyllum, E. vanderwerffii, E. vareschianum, andE. variolatum.     

Repression of the integrated papillomavirus E6/E7 promoter is required for growth suppression of cervical cancer cells

The human papillomavirus (HPV) E2 protein is an important regulator of viral E6 and E7 gene expression. E2 can repress the viral promoter for E6 and E7 expression as well as block progression of the cell cycle in cancer cells harboring the DNA of "high-risk" HPV types. Although the phenomenon of E2-mediated growth arrest of HeLa cells and other HPV-positive cancer cells has been well documented, the specific mechanism by which E2 affects cellular proliferation has not yet been elucidated. Here, we show that bovine papillomavirus (BPV) E2-induced growth arrest of HeLa cells requires the repression of the E6 and E7 promoter. This repression is specific for E2TA and not E2TR, a BPV E2 variant that lacks the N-terminal transactivation domain. We demonstrate that expression of HPV16 E6 and E7 from a heterologous promoter that is not regulated by E2 rescues HeLa cells from E2-mediated growth arrest. Our data indicate that the pathway of E2-mediated growth arrest of HeLa cells requires repression of E6 and E7 expression through an activity specified by the transactivation domain of E2TA.     

Solution structure determination and mutational analysis of the papillomavirus E6 interacting peptide of E6AP

E6AP is a cellular protein that binds cancer-related papillomaviral E6 proteins. The E6 binding domain, called E6ap, is located on an 18-amino acid segment of E6AP. The corresponding peptide was synthesized and its structure determined by nuclear magnetic resonance spectroscopy. The overall structure of the peptide is helical. A consensus E6-binding sequence among different E6 interacting proteins contains three conserved hydrophobic residues. In the structure of the E6AP peptide, the three conserved leucines (Leu 9, Leu 12, and Leu 13) form a hydrophobic patch on one face of the alpha-helix. Substitution of any of these leucines with alanine abolished binding to E6 protein, indicating that the entire hydrophobic patch is necessary. Mutation of a glutamate to proline, but not alanine, also disrupted the interaction between E6 and E6AP protein, suggesting that the E6-binding motif of the E6AP protein must be helical when bound to E6. Comparison of the E6ap structure and mutational results with those of another E6-binding protein (E6BP/ERC-55) indicates the existence of a general E6-binding motif.     

Human papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligase

The E6 protein of the high-risk human papillomaviruses (HPVs) and the cellular ubiquitin-protein ligase E6AP form a complex which causes the ubiquitination and degradation of p53. We show here that HPV16 E6 promotes the ubiquitination and degradation of E6AP itself. The half-life of E6AP is shorter in HPV-positive cervical cancer cells than in HPV-negative cervical cancer cells, and E6AP is stabilized in HPV-positive cancer cells when expression of the viral oncoproteins is repressed. Expression of HPV16 E6 in cells results in a threefold decrease in the half-life of transfected E6AP. E6-mediated degradation of E6AP requires (i) the binding of E6 to E6AP, (ii) the catalytic activity of E6AP, and (iii) activity of the 26S proteasome, suggesting that E6-E6AP interaction results in E6AP self-ubiquitination and degradation. In addition, both in vitro and in vivo experiments indicate that E6AP self-ubiquitination results primarily from an intramolecular transfer of ubiquitin from the active-site cysteine to one or more lysine residues; however, intermolecular transfer can also occur in the context of an E6-mediated E6AP multimer. Finally, we demonstrate that an E6 mutant that is able to immortalize human mammary epithelial cells but is unable to degrade p53 retains its ability to bind and degrade E6AP, raising the possibility that E6-mediated degradation of E6AP contributes to its ability to transform mammalian cells.     

Purification partielle et propriétés des amylases de la poire

Three amylases E1, E2 and E3 have been extracted from Passe-Crassane pears pulp and partially purified. A good extraction of these enzymes is obtained only with non ionic detergents. Furthermore, solubilization of amylase E1 needs the use of these compounds. Separation of the 3 amylases is obtained by chromatography on Sephadex G100. E1 and E2 are β-amylases whereas E3 is an α-amylase. The first two have similar kinetic properties and interconversion of one form into the other is possible. It is suggested that the insoluble form E1 is the result of the association of β-amylase E2 with other compounds present in the fruit.     

根据形态和叶片微形态特征讨论无芒披碱草的归并

通过形态学观测和叶片解剖特征分析,比较了无芒披碱草、短芒披碱草及老芒麦3个近缘种的主要性状差异,以探讨无芒披碱草的系统分类归属.结果表明,在外部形态上无芒披碱草与短芒披碱草差异甚小,难以进行区分,但与老芒麦差异明显,是典型的种间关系;在叶片解剖上,无芒披碱草的绝大多数特征与短芒披碱草的一致或类同,可与老芒麦的却存在明显间断.故研究认为:无芒披碱草与短芒披碱草是同一个种,无芒披碱草应作为短芒披碱草的异名.     

How dihydrolipoamide dehydrogenase-binding protein binds dihydrolipoamide dehydrogenase in the human pyruvate dehydrogenase complex

The dihydrolipoamide dehydrogenase-binding protein (E3BP) and the dihydrolipoamide acetyltransferase (E2) component enzyme form the structural core of the human pyruvate dehydrogenase complex by providing the binding sites for two other component proteins, dihydrolipoamide dehydrogenase (E3) and pyruvate dehydrogenase (E1), as well as pyruvate dehydrogenase kinases and phosphatases. Despite a high similarity between the primary structures of E3BP and E2, the E3-binding domain of human E3BP is highly specific to human E3, whereas the E1-binding domain of human E2 is highly specific to human E1. In this study, we characterized binding of human E3 to the E3-binding domain of E3BP by x-ray crystallography at 2.6-angstroms resolution, and we used this structural information to interpret the specificity for selective binding. Two subunits of E3 form a single recognition site for the E3-binding domain of E3BP through their hydrophobic interface. The hydrophobic residues Pro133, Pro154, and Ile157 in the E3-binding domain of E3BP insert themselves into the surface of both E3 polypeptide chains. Numerous ionic and hydrogen bonds between the residues of three interacting polypeptide chains adjacent to the central hydrophobic patch add to the stability of the subcomplex. The specificity of pairing for human E3BP with E3 is interpreted from its subcomplex structure to be most likely due to conformational rigidity of the binding fragment of the E3-binding domain of E3BP and its exquisite amino acid match with the E3 target interface.     

The Alphavirus E3 Glycoprotein Functions in a Clade-Specific Manner

The 80 trimeric, glycoprotein spikes that cover the surface of alphavirus particles are required for mediating viral entry into a host cell. Spike assembly is a regulated process that requires interactions between five structural proteins, E3, E2, 6K and its translational frameshift product TF, and E1. E3 is a small, ∼65-amino-acid glycoprotein that has two known functions: E3 serves as the signal sequence for translocation of the E3-E2-6K-E1 polyprotein into the endoplasmic reticulum (ER), and cleavage of E3 from E2 is essential for virus maturation. Nonetheless, when E3 is replaced with an ER signal sequence, spikes do not form and infectious particles are not assembled, suggesting an additional role(s) for E3 in the viral life cycle. To further investigate the role of E3 in spike assembly, we made chimeric viruses in which E3 from one alphavirus species is replaced with E3 from another species. Our results demonstrate that when E3 is interchanged between alphavirus species that belong to the same virus clade, viral titers and particle morphologies and compositions are similar to what are observed for the parental virus. In contrast, for chimeras in which E3 is derived from a different clade than the parental virus, we observed reduced titers and the formation of particles with atypical morphologies and protein compositions. We further characterized the E3 chimeras using a combination of structure-function and revertant analyses. This work revealed two specific interactions between E3 and its cognate E2 glycoprotein that are important for regulating spike assembly.     

Subunit binding in the pyruvate dehydrogenase complex from bovine kidney and heart

Binding of pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3) to the isolated dihydrolipoamide acetyltransferase (E2) core of the pyruvate dehydrogenase complex from bovine heart and kidney was investigated with equilibrium, competitive binding, and kinetic methods. E2, which consists of 60 subunits arranged with icosahedral 532 symmetry, apparently possesses six equivalent, noninteracting binding sites for E3 dimers. It is proposed that each E3 dimer extends across 2 of the 12 faces of the E2 pentagonal dodecahedron. The equilibrium constant (Kd) for dissociation of E3 from E2 is about 3 nM, and the dissociation rate constant is about 0.057 min-1. For E1, Kd is about 13 nM, and the dissociation rate constant is about 0.043 min-1. Extensive phosphorylation of E1 (about three phosphoryl groups per E1 tetramer) increases Kd to about 40 nM.