B-cell- and myocyte-specific E2-box-binding factors contain E12/E47-like subunits.

Recent studies have identified a family of DNA-binding proteins that share a common DNA-binding and dimerization domain with the potential to form a helix-loop-helix (HLH) structure. Various HLH proteins can form heterodimers that bind to a common DNA sequence, termed the E2-box. We demonstrate here that E2-box-binding B-cell- and myocyte-specific nuclear factors contain subunits which are identical or closely related to ubiquitously expressed (E12/E47) HLH proteins. These biochemical function for E12/E47-like molecules in mammalian differentiation, similar to the genetically defined function of daughterless in Drosophila development.     

Negative regulation of the bovine papillomavirus E5, E6, and E7 oncogenes by the viral E1 and E2 genes.

Papillomaviruses induce benign squamous epithelial lesions that infrequently are associated with uncontrolled growth or malignant conversion. The virus-encoded oncogenes are clearly under negative regulation since papillomaviruses can latently infect cells and since different levels of viral oncogene expression are seen within the layers of differentiating infected epitheliomas. We used bovine papillomavirus type 1 (BPV-1) to investigate the mechanisms involved in the negative regulation of transformation. We found that the following two distinct and interacting mechanisms negatively regulate BPV-1 transformation effected by virally encoded trans-acting factors: (i) E2 repressors suppress transformation by the E6 and E7 oncogenes, and (ii) E1 and the E2 transactivator suppress transformation by the E6, E7, and E5 oncogenes. These systems interact in that the E2 repressors function to relieve the transformation suppression effected by the E1 and E2 transactivator genes. A BPV-1 mutant that lacked E2 repressors and E1 had greatly augmented transformation capacity. Analysis of this mutant revealed that the enhanced transformation was due to expression of the E6 and E7 genes in the absence of E5, revealing a previously unappreciated potency and synergy for the BPV-1 E6 and E7 oncogenes.     

Differentiation and identification of Enterococcus durans, E. hirae and E. villorum

AIMS: To compare different tests in the identification of Enterococcus durans, E. hirae and E. villorum strains. These bacteria belong to the E. faecium species group and are phylogenetically closely related, as evidenced by 16S rRNA sequence homologies of over 98.8%. METHODS AND RESULTS: Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of whole-cell protein, tRNA interpacer polymerase chain reaction (PCR) and arbitrarily-primed (D11344-primed AP) -PCR analysis correctly identified all three species in a collection of strains from very diverse origins. In contrast, biochemical reactions only allowed the unequivocal differentiation of the three species as a group from the other enterococci. Within this group, D-xylose acidification can be used to differentiate E. villorum, but exceptions occur. Strains highly susceptible to clindamycin can be identified as E. durans, but many strains of this species cannot be differentiated from E. hirae and E. villorum due to acquired resistance. CONCLUSIONS: Despite their close relationship, E. durans, E. hirae and E. villorum can be differentiated by genomic methods and by whole-cell protein analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Only a minority of strains of these three enterococcal species can be identified reliably by the currently available and commonly applied phenotypic tests.     

Apolipoprotein E and atherosclerosis

Apolipoprotein E plays a key protective role in atherosclerosis. Its capacity to safeguard against this disease can be attributed to at least three distinct functions. First, plasma apolipoprotein E maintains overall plasma cholesterol homeostasis by facilitating efficient hepatic uptake of lipoprotein remnants. Second, lesion apolipoprotein E in concert with apolipoprotein A-I facilitates cellular cholesterol efflux from macrophage foam cells within the intima of the lesion. Third, lesion apolipoprotein E directly modifies both macrophage- and T lymphocyte-mediated immune responses that contribute to this chronic inflammatory disease.     

Purification and application of bacterially expressed chimeric protein E1E2 of hepatitis C virus

E1 and E2 glycoproteins are structural components of hepatitis C virus (HCV) virion. They are involved in cellular receptors interaction, neutralising antibodies elicitation, and viral morphogenesis. They are considered as major candidates for anti-HCV vaccine. In this report, we first expressed tandem E1E2 as well as C-terminally truncated E1 fragment and C-terminally truncated E2 fragment, respectively, in Escherichia coli cells and the proteins were purified to homogenesis. All the purified proteins can react specifically with patient sera. Both purified chimeric protein E1E2 and protein E2 can interact with a putative cellular receptor CD81, while purified protein E1 cannot interact with CD81. The sera of rabbit immunized with the E1E2 inhibited the binding of E2 protein to the major extracellular loop of human CD81 and reacted with both proteins E1 and E2, respectively. Anti-E1 and E2 antibodies can be generated simultaneously in the rabbit immunized with the E1E2, and the titers of antibodies were 63 or 56% higher than the titers induced by E1 or E2 alone, respectively. The results suggest that E1 and E2 can enhance their immunogenicity each other in chimeric protein E1E2 and the E. coli-derived chimeric protein E1E2 and corresponding antisera can be used as an useful tools in anti-HCV vaccine research.     

Ubiquitination without E3

In the current issue of Molecular Cell, Hoeller et al. (2007) demonstrated that proteins bearing ubiquitin-binding domains (UBDs) can be ubiquitinated directly by E2-conjugating enzymes, bypassing the requirement for E3 ubiquitin ligases.     

Production and Characterization of Improved Adenovirus Vectors with the E1, E2b, and E3 Genes Deleted

Adenovirus (Ad)-based vectors have great potential for use in the gene therapy of multiple diseases, both genetic and nongenetic. While capable of transducing both dividing and quiescent cells efficiently, Ad vectors have been limited by a number of problems. Most Ad vectors are engineered such that a transgene replaces the Ad E1a, E1b, and E3 genes; subsequently the replication-defective vector can be propagated only in human 293 cells that supply the deleted E1 gene functions in trans. Unfortunately, the use of high titers of E1-deleted vectors has been repeatedly demonstrated to result in low-level expression of viral genes still resident in the vector. In addition, the generation of replication-competent Ad (RCA) by recombination events with the E1 sequences residing in 293 cells further limits the usefulness of E1-deleted Ad vectors. We addressed these problems by isolating new Ad vectors deleted for the E1, E3, and the E2b gene functions. The new vectors can be readily grown to high titers and have several improvements, including an increased carrying capacity and a theoretically decreased risk for generating RCA. We have also demonstrated that the further block to Ad vector replication afforded by the deletion of both the E1 and E2b genes significantly diminished Ad late gene expression in comparison to a conventional E1-deleted vector, without destabilization of the modified vector genome. The results suggested that these modified vectors may be very useful both for in vitro and in vivo gene therapy applications.     

Vitamin E and oxidative stress

Oxidative stress can result from or be enhanced by a large variety of conditions, including nutritional imbalance, exposure to chemical and physical agents in the environment, strenuous physical activities, injury, and hereditary disorders. While many enzymes and compounds are involved in protecting cells from the adverse effects of oxidative stress, vitamin E occupies an important and unique position in the overall antioxidant defense. The antioxidant function of vitamin E is closely related to the status of many dietary components. Vitamin E-depleted animals are generally more susceptible to the adverse effects of environmental agents than supplemented animals. Also, vitamin E supplementation is beneficial to certain groups of the population. However, supplementing vitamin E in experimental subjects maintained on a nutritionally adequate diet does not always provide additional protection. Differential metabolic responses in various organs and differences in experimental conditions often contribute in the discrepancies in the literature. The lack of clear evidence for the occurrence of lipid peroxidation or antioxidant function of vitamin E in vivo can be attributed partly to the presence of active pathways for metabolizing hydroperoxides, aldehydes, and other oxidation products. Specific and sensitive techniques for measuring lipid peroxidation products in biological systems are essential for understanding the role of free radical-induced lipid peroxidation in tissue damage and antioxidant function of vitamin E in vivo.     

Hepatitis E: an overview

The hepatitis E virus (HEV) is a non-enveloped, positive-sense, single-stranded RNA virus with icosahedral symmetry. Although it is related to the alpha-virus superfamily, the HEV is classified as a separate Hepatitis E-like viruses genus. Infection in humans occurs in sporadic and epidemic forms and can cause an acute, self-limited, icteric hepatitis. Recent studies indicate the existence of a reservoir in animals.     

Leaky scanning is the predominant mechanism for translation of human papillomavirus type 16 E7 oncoprotein from E6/E7 bicistronic mRNA

Human papillomaviruses (HPV) are unique in that they generate mRNAs that apparently can express multiple proteins from tandemly arranged open reading frames. The mechanisms by which this is achieved are uncertain and are at odds with the basic predictions of the scanning model for translation initiation. We investigated the unorthodox mechanism by which the E6 and E7 oncoproteins from human papillomavirus type 16 (HPV-16) can be translated from a single, bicistronic mRNA. The short E6 5' untranslated region (UTR) was shown to promote translation as efficiently as a UTR from Xenopus beta-globin. Insertion of a secondary structural element into the UTR inhibited both E6 and E7 expression, suggesting that E7 expression depends on ribosomal scanning from the 5' end of the mRNA. E7 translation was found to be cap dependent, but E6 was more dependent on capping and eIF4F activity than E7. Insertion of secondary structural elements at various points in the region upstream of E7 profoundly inhibited translation, indicating that scanning was probably continuous. Insertion of the E6 region between Renilla and firefly luciferase genes revealed little or no internal ribosomal entry site activity. However when E6 was located at the 5' end of the mRNA, it permitted over 100-fold-higher levels of downstream cistron translation than did the Renilla open reading frame. Internal AUGs in the E6 region with strong or intermediate Kozak sequence contexts were unable to inhibit E7 translation, but initiation at the E7 AUG was efficient and accurate. These data support a model in which E7 translation is facilitated by an extreme degree of leaky scanning, requiring the negotiation of 13 upstream AUGs. Ribosomal initiation complexes which fail to initiate at the E6 start codon can scan through to the E7 AUG without initiating translation, but competence to initiate is achieved once the E7 AUG is reached. These findings suggest that the E6 region of HPV-16 comprises features that sponsor both translation of the E6 protein and enhancement of translation at a downstream site.     

Functional interactions between papillomavirus E1 and E2 proteins.

DNA replication of papillomaviruses requires the viral E1 and E2 proteins. These proteins bind cooperatively to the viral origin of replication (ori), which contains binding sites for both proteins, forming an E1-E2-ori complex which is essential for initiation of DNA replication. To map the domains in E2 that are involved in the interaction with E1, we have used chimeric bovine papillomavirus (BPV)/human papillomavirus type 11 (HPV-11) E2 proteins. The results from this study show that both the DNA binding domain and the transactivation domain from BPV E2 independently can interact with BPV E1. However, the roles of these two interactions are different: the interaction between E1 and the activation domain of E2 is necessary and sufficient for cooperativity in binding and for DNA replication; the interaction between E1 and the DNA binding domain of E2 is required only when the binding sites for E1 and E2 are adjacent to each other, and the function of this interaction appears to be to facilitate the interaction between E1 and the transactivation domain of E2. These results indicate that the cooperative binding of E1 and E2 to the BPV ori takes place via a novel two-stage mechanism where one interaction serves as a trigger for the formation of the second, productive, interaction between the two proteins.     

Vesicular stomatitis virus-based therapeutic vaccination targeted to the E1, E2, E6, and E7 proteins of cottontail rabbit papillomavirus

Persistent human papillomavirus (HPV)-associated benign and malignant lesions are a major cause of morbidity and mortality worldwide. Vaccination against HPV early proteins could provide an effective means of treating individuals with established infections. Recombinant vesicular stomatitis virus (VSV) vectors have been used previously to elicit strong humoral and cellular immune responses and develop prophylactic vaccines. We have shown that VSV vectors also can be used to elicit therapeutic immunity in the cottontail rabbit papillomavirus (CRPV)-rabbit model of high-risk HPV infection. In the present study, three new VSV vectors expressing the CRPV E1, E2, or E7 protein were produced and compared to the previously generated VSV-E6 vector for therapeutic efficacy. To determine whether vaccine efficacy could be augmented by simultaneous vaccination against two CRPV proteins, the four vaccines were delivered individually and in all possible pairings to rabbits 1 week after CRPV infection. Control rabbits received the recombinant wild-type VSV vector or medium only. Cumulative papilloma volumes were computed for analysis of the data. The analyses showed that VSV-based vaccination against the E1, E2, E6, or E7 protein significantly reduced papilloma volumes relative to those of the controls. Furthermore, VSV-based CRPV vaccination cured all of the papillomas in 5 of 30 rabbits. Of the individual vaccines, VSV-E7 was the most effective. The VSV-E7 vaccine alone was the most effective, as it reduced cumulative papilloma volumes by 96.9% overall, relative to those of the controls, and ultimately eliminated all of the disease in all of the vaccinees. Vaccine pairing was not, however, found to be beneficial, suggesting antigenic competition between the coexpressed CRPV proteins. These preclinical results, obtained in a physiologically relevant animal model of HPV infection, demonstrate that VSV vectors deserve serious consideration for further development as therapeutic antitumor vaccines.     

Mechanism of export of colicin E1 and colicin E3.

The mechanism of export of colicins E1 and E3 was examined. Neither colicin E1, colicin E3, Nor colicin E3 immunity protein appears to be synthesized as a precursor protein with an amino-terminal extension. Instead, the colicins, as well as the colicin E3 immunity protein, appear to leave the cells where they are made, long after their synthesis, by a nonspecific mechanism which results in increased permeability of the producing cells. Induction of ColE3-containing cells with mitomycin C leads to actual lysis of those cells, as some time after synthesis of the colicin E3 and its immunity protein has been completed. Induction of ColE1-containing cells results in increased permeability of the cells, but not in actual lysis, and most of the colicin E1 produced never leaves the producing cells. Intracellular proteins such as elongation factor G can be found outside of colicinogenic cells after mitomycin C induction, along with the colicin. Until substantial increases in permeability occur, most of the colicin remains cell associated, in the soluble cytosol, rather than in a membrane-associated form.     

Comparative study of the antimutagenic potential of Vitamin E in different E. coli strains

The antimutagenic potential of Vitamin E due to its antioxidative properties was studied. The new Escherichia coli K12 assay-system designed in our laboratory was employed in order to detect the antimutagenic potential of Vitamin E and to determine its molecular mechanisms of action. The assay is composed of three tests. In Test A, we examine the influence of the antioxidant on induced oxidative mutagenesis in a repair-proficient strain. Spontaneous mutagenesis is monitored in Test B, which is performed with two mutator strains, one mismatch repair-deficient (mutS) and another deficient in 8-oxo-dGTP-ase activity (mutT). In Test M, a repair-proficient strain and its mismatch repair-deficient counterpart (mutH), both carrying a plasmid with microsatellite sequences, are used to measure the level of microsatellite instability. To examine the antimutagenic potential of Vitamin E we also used the WP2 antimutagenicity test. Protective properties of Vitamin E against oxidative mutagenesis were detected in all tests with the E. coli K12 assay-system as well as in the WP2 antimutagenicity test. This study confirms that mismatch repair is essential for repair of oxidative DNA damage. The results obtained indicate that Vitamin E prevents the formation of DNA adducts by lipid peroxidation products rather than those formed by direct oxidation of DNA bases. Moreover, it can reduce microsatellite instability. After further validation, the new E. coli K12 assay-system can be used to test the antimutagenic potential of antioxidants.     

Processing of Procarboxypeptidase E into Carboxypeptidase E Occurs in Secretory Vesicles

Abstract: Carboxypeptidase E (CPE) functions in the posttranslational processing of bioactive peptides. Like other peptide processing enzymes, CPE is initially produced as a precursor ("proCPE") that undergoes posttranslational processing at a site containing five adjacent Arg residues near the N-terminus and at other sites near the C-terminus of proCPE. The time course of the N-terminal processing step suggests that this conversion occurs in either the Golgi apparatus or the secretory vesicles. To delineate further the site of proCPE processing, pulse/chase analysis was performed under conditions that block transit out of the Golgi apparatus (brefeldin A, carbonyl cyanide m -chlorophenylhydrazone, or 20°C) or that block acidification of vesicles (chloroquine, monensin, or ammonium chloride). The results of these analysis suggest that efficient proCPE processing requires an acidic post-Golgi compartment. To test whether known processing enzymes can perform this cleavage, purified proCPE was incubated with furin, prohormone convertase 1, or a dynorphin converting enzyme, and the products were analyzed on denaturing polyacrylamide gels. Furin cleaves proCPE within the N-terminal region, although the reaction is not very efficient, requiring relatively large amounts of furin or long incubation times. The other two peptide processing enzymes did not cleave proCPE, whereas a relatively small amount of secretory granule extract was able to convert proCPE into CPE. Taken together, these findings suggest that the conversion of proCPE into CPE occurs primarily in secretory vesicles.