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E J Androphy

Publications and source records attributed to E J Androphy.

At least 19 recordsLinked to original sources
PubMed1997

Sequences flanking the core DNA-binding domain of bovine papillomavirus type 1 E2 contribute to DNA-binding function.

We have compared a series of molecular constructs that contain the minimal DNA-binding and dimerization domain of bovine papillomavirus type 1 (BPV-1) E2 alone or this binding domain plus the adjacent 16 or 40 amino acids to test the role of the flanking sequences in E2 function. The presence of these sequences resulted in an up to eightfold increase in the affinity of E2 for its target DNA and stabilized the protein against denaturation both in the absence of DNA and in the form of DNA-protein complexes. In addition, an aspartic acid-to-tyrosine mutation within the flanking region blocked DNA binding and function. These data demonstrate that sequences flanking the core domain contribute to E2 function and are, in fact, an integral part of the DNA-binding domain of BPV-1 E2.

Animals↗
PubMed1996

Genetic analysis of the bovine papillomavirus E2 transcriptional activation domain.

The bovine papillomavirus type 1 E2 transactivator has a large amino-terminal 215-residue transcriptional activation domain (TAD) that is active in Saccharomyces cerevisiae and higher eukaryotic cells. Comparison to other transcriptional activators suggests that its functions may be mediated in part through two acidic regions, A1 and A2, in this domain. We have characterized the functional elements within the E2 TAD using LexA-E2 fusions and by screening randomly generated libraries of E2 mutations for transcriptional activation in yeast. The A1 region was highly sensitive to substitutions that reduce negative charge, although there was not a perfect correlation between overall charge and transcriptional activity. Mutations were isolated within a hydrophobic amino acid motif that overlaps the A2 region and resembles elements described in other viral and cellular transactivation domains. When fused to the LexA DNA binding domain, this hydrophobic motif within the acidic A2 region was unable to activate transcription in S. cerevisiae. Multiple highly defective mutations primarily altering hydrophobic amino acids were identified in the distal third of the E2 TAD. The transcription phenotype of many of these E2 TAD mutations was similar in yeast and COS cells.

Amino Acid Sequence↗
PubMed1996

The BPV-1 E2 DNA-contact helix cysteine is required for transcriptional activation but not replication in mammalian cells.

The papillomavirus E2 protein contains an amino-terminal region thought necessary and sufficient to support transcriptional activation and a carboxy-terminal region shown to direct sequence-specific DNA binding and dimerization. A cysteine residue in the center of the E2 DNA recognition helix is highly conserved among papillomavirus E2 proteins. Mutations of this cysteine in bovine papillomavirus type 1 E2 to serine and glycine resulted in proteins which failed to activate E2-dependent promoters in mammalian cells. These E2 mutants were DNA-binding competent, dimeric, and nuclear. When fused to the VP16 transactivation domain, C-terminal regions of E2 containing the mutations at 340 supported transcriptional activation, indicating that the heterologous trans-activation domain did not require cysteine in the DNA-binding helix as did the full-length E2 transactivating protein. Although cysteine-340 was required for transcriptional activation it was not required for DNA replication in vivo. Together, these results suggest that the E2 DNA-binding domain may directly contribute to functions of transcriptional activation previously thought limited to the N-terminal domain.

3T3 Cells↗
PubMed1996

Transcriptional activation function is not required for stimulation of DNA replication by bovine papillomavirus type 1 E2.

Bovine papillomavirus type 1 replication was previously shown to require both the E1 initiator protein and the E2 transactivator protein. We show here that E1, in the absence of E2, is sufficient for low-level bovine papillomavirus type 1 DNA replication in C-33A cells. In addition, studies of genetically isolated E2 point mutants demonstrate that enhancement of replication by E2 does not require its transcriptional activation function. The uncoupling of the E2 functions suggests that stimulation of transcription and replication by enhancer proteins occurs via divergent mechanisms.

Animals↗
PubMed1996

Mutational analysis of human papillomavirus type 16 E6 demonstrates that p53 degradation is necessary for immortalization of mammary epithelial cells.

We have previously demonstrated that normal human mammary epithelial cells (MECs) are efficiently immortalized by human papillomavirus type 16 (HPV16) E6. HPV16 E6 binds to and induces p53 degradation in vitro and induces a marked reduction of p53 protein in MECs. Low-risk HPV6 E6 is defective for p53 binding and degradation in vitro but immortalized MECs at low efficiency. The HPV6 E6-immortalized MECs had markedly reduced levels of p53. To directly investigate whether the ability of HPV16 E6 to stimulate p53 degradation is required for E6-induced immortalization, a series of HPV16 E6 mutants were analyzed for the ability to bind and degrade p53 in vitro, induce a reduction in p53 levels in vivo, and immortalize MECs. We observed that one set of mutants efficiently immortalized MECs, caused a reduction in p53 levels in vivo, and degraded p53 in vitro. Other mutants immortalized MECs with low efficiency and either induced p53 degradation at low levels or were unable to induce p53 degradation in vitro; however, all of the immortal clones displayed low levels of p53. A third class of mutants did not immortalize MECs and failed to induce a reduction in p53 levels in vivo or degrade p53 in vitro. These results demonstrate that a reduction in p53 protein levels due to enhanced degradation is essential for MEC immortalization by HPV16 E6.

Base Sequence↗
PubMed1995

Interaction of papillomavirus E6 oncoproteins with a putative calcium-binding protein.

Human papillomaviruses (HPVs) are associated with the majority of cervical cancers and encode a transforming protein, E6, that interacts with the tumor suppressor protein p53. Because E6 has p53-independent transforming activity, the yeast two-hybrid system was used to search for other E6-binding proteins. One such protein, E6BP, interacted with cancer-associated HPV E6 and with bovine papillomavirus type 1 (BPV-1) E6. The transforming activity of BPV-1 E6 mutants correlated with their E6BP-binding ability. E6BP is identical to a putative calcium-binding protein, ERC-55, that appears to be localized in the endoplasmic reticulum.

Bovine papillomavirus 1↗
PubMed1995

The domain of p53 required for binding HPV 16 E6 is separable from the degradation domain.

The E6 proteins of specific cancer-associated human papillomaviruses (HPVs) complex with and mediate degradation of the cellular anti-oncogene p53 in vitro. A critical property of p53 is its ability to stimulate transcription from promoters containing its recognition sequence. HPV E6, mutant p53 proteins, and several DNA tumor virus oncogenes inhibit the transcriptional activity of wild-type p53. In this report, the structural requirements for the interaction between HPV 16 E6 and p53 were examined both in vivo and in vitro. p53-stimulated transcription was efficiently inhibited by wild-type HPV 16 E6 and E6 mutants competent for p53 binding and degradation. A series of p53 deletions and hybrid proteins with heterologous DNA binding, dimerization and transactivation domains were analysed for transcriptional interaction with HPV 16 E6 to determine the domains of p53 required for transcriptional inhibition. These chimeric proteins were also analysed for E6 binding and E6-mediated degradation in vitro. In both assays, complex formation with E6 was mediated through the amino-terminal 345 amino acids of p53 without a specific requirement for its C-terminus. Hybrid proteins containing residues 161-345 of p53 also bound E6, but this segment of p53 was not susceptible to E6 induced proteolysis. A second region of p53, within its N-terminal 160 aa, is required for E6 induced degradation of complexed p53. Taken together, these results suggest that the complex formation between E6 and p53 is not mediated through the C-terminus of p53 and that binding and degradation are separable.

Animals↗
PubMed1995

A novel method for selective isotope labeling of bacterially expressed proteins.

A novel method for isotope labeling in selected amino acids is presented for use with the T7 RNA polymerase system. The protocol is illustrated with the DNA-binding domain from the E2 protein of bovine papillomavirus, BPV-1. On addition of rifampicin, protein expression occurs exclusively from the gene controlled by the T7 promoter. Since the bacteria are now dedicated to the production of E2 protein, labeling with specific amino acids is efficiently performed. For example, 10 mg/l of 15N-labeled phenylalanine is shown to be sufficient for incorporation of the label, without scrambling, and without the use of an auxotrophic strain.

DNA-Binding Proteins↗
PubMed1994

Isolation, sequence analysis and characterization of a cDNA encoding human chaperonin 10.

A full-length cDNA clone encoding chaperonin 10 (cpn10) from a HeLa cell cDNA library was isolated. The cDNA is 538 bp in length, contains an ATG codon and a putative polyadenylation signal, and specifies a protein of 102 amino acids. Immunoprecipitation experiment showed that this human cpn10 has an apparent molecular mass of 11 kDa in sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Amino Acid Sequence↗
PubMed1994

Specific inhibition of a human papillomavirus E2 trans-activator by intracellular delivery of its repressor.

Papillomaviruses are the causative agents of benign and malignant epithelial tumors of the skin and mucosa. They encode a DNA-binding protein, E2, that regulates viral transcription and replication, making it an important therapeutic target. By deleting the amino-terminal trans-activation domain of human papillomavirus type 16 (HPV-16) E2 while retaining its carboxy-terminal DNA binding and dimerization domain, an E2 repressor (E2R) that efficiently inhibits transcriptional activation by full-length HPV E2 was generated. To deliver this repressor protein into animal cells, we have utilized the human immunodeficiency virus type 1 (HIV-1) Tat protein which itself is taken up efficiently into intact cells. Chimeras of E2R and the cellular uptake domain of Tat specifically inhibited E2-dependent reporter gene expression in COS-7 cells. Treatment of cervical intraepithelial neoplasia cells having episomally replicating HPV-31 DNA with this Tat-E2R protein led to a dose-dependent loss of HPV DNA copies and inhibition of cell growth. Tat-mediated delivery can be a valuable tool for assessing protein function and may allow the development of novel therapeutic proteins having intracellular targets.

Amino Acid Sequence↗
PubMed1993

Enhanced degradation of p53 protein in HPV-6 and BPV-1 E6-immortalized human mammary epithelial cells.

Normal mammary epithelial cells are efficiently immortalized by the E6 gene of human papillomavirus (HPV)-16, a virus commonly associated with cervical cancers. Surprisingly, introduction of the E6 gene from HPV-6, which is rarely found in cervical cancer, or bovine papillomavirus (BPV)-1, into normal mammary cells resulted in the generation of immortal cell lines. The establishment of HPV-6 and BPV-1 E6-immortalized cells was less efficient and required a longer period in comparison to HPV-16 E6. These HPV-6- and BPV-1 E6-immortalized cells demonstrated dramatically reduced levels of p53 protein by immunoprecipitation. While the half-life of p53 protein in normal mammary epithelial cells was approximately 3 h, it was reduced to approximately 15 min in all the E6-immortalized cells. These results demonstrate that the E6 genes of both high-risk and low-risk papilloma viruses immortalize human mammary epithelial cells and induce a marked degradation of p53 protein in vivo.

Base Sequence↗
PubMed1993

The tryptophan bridge is a critical feature of the papillomavirus E2 DNA binding domain.

The papillomavirus E2 protein is a DNA binding protein that regulates viral transcription and replication. E2 binds DNA as a dimer. Recent crystallographic data for E2 complexed to DNA revealed that novel peptide structures in E2 mediated dimerization and DNA binding. To identify important features of these motifs we have used limited proteolysis and urea denaturation as biochemical probes for structure, applying these techniques to E2 alone, E2 bound to DNA, cross-linked products, and mutants that were targeted at Trp360, a contact point along the dimer interface. DNA binding stabilized E2 structure, shifting the point at which it denatures from 5 to 7.6 M urea. In contrast, Trp360 mutant proteins, while dimeric, were more sensitive to denaturation by urea when bound to DNA. The most striking results came from uv cross-linking studies in which Trp360 was targeted as the site of cross-linking. Ultraviolet cross-linking dramatically increased the resistance of E2 to proteolysis regardless of the protease tested and with no deleterious effect on the affinity of E2 for DNA. Cross-linking through Cys356 with bismaleimidohexane did not promote stabilization. The ability to stabilize or destabilize E2 by Trp360-targeted modifications demonstrates the importance of the Trp360-Trp360 interaction, which may represent a general feature of the beta-barrel motif.

Base Sequence↗
PubMed1992

Amino acids necessary for DNA contact and dimerization imply novel motifs in the papillomavirus E2 trans-activator.

The bovine papillomavirus E2 protein regulates viral transcription by binding as a dimer to the DNA sequence ACCGN4CGGT. The dimerization and DNA-binding properties are localized within its carboxy-terminal 85 amino acids (325-410). Utilizing random mutagenesis coupled with phenotypic selection in yeast, functionally important amino acids in the DNA-binding domain were identified. Four trans-activation defective point mutants within a short segment (amino acids 337-344) were DNA binding defective but dimeric. The mutation of a conserved tryptophan to serine also eliminated DNA binding, but loss of dimerization was implicated because addition of dimeric monoclonal antibody complemented this defect. A simple assay for E2 dimerization was developed using UV irradiation to produce an interchain cross-link within a dimer. No heterodimeric complexes were formed when pools of E2 of varying lengths were mixed, and only proteins with tryptophan at position 360 could be UV cross-linked. Peptide mapping of irradiated E2 protein localized the cross-link to an 18-amino-acid region bracketing this tryptophan. Substitutions for this tryptophan demonstrated the requirement for a hydrophobic residue at this position, but surprisingly, even alanine was functional. Replacement of this tryptophan with three polar amino acids or glycine eliminated DNA-binding activity, but addition of dimeric monoclonal antibody restored this function. The amino acids that were identified as being involved in DNA contact and dimerization imply that these functions are mediated by novel binding motifs.

Amino Acid Sequence↗
PubMed1992

Mechanism of action of the papillomavirus E2 repressor: repression in the absence of DNA binding.

Repression of papillomavirus E2-dependent gene expression was studied by using transient transfections into mouse embryo fibroblast cells. Cotransfection of a gene corresponding to the naturally occurring repressor E2-TR along with the full-length E2 gene resulted in up to 98% repression of E2-dependent reporter gene expression. A series of E2 DNA-binding domain mutants were transferred into the E2-TR form and characterized for their ability to repress E2-dependent transactivation. All mutants which were defective for DNA binding but were dimerization competent repressed E2 transactivation as well or nearly as well as the wild-type repressor. E2 mutants which lacked dimerization activity repressed transactivation poorly or not at all. These results indicate that the E2 repressor can inhibit transcription, in the absence of DNA binding, by forming heterodimers with full-length E2.

Amino Acid Sequence↗
PubMed1991

Cooperative binding of the E2 protein of bovine papillomavirus to adjacent E2-responsive sequences.

The DNA-binding properties of purified full-length E2 protein from bovine papillomavirus type 1 have been investigated by utilizing a quantitative gel shift analysis. By using a recombinant baculovirus which express the E2 open reading frame from the polyhedrin promoter, the full-length E2 protein was synthesized in insect cells and purified to homogeneity by using an E2 binding site (ACCGN4CGGT)-specific oligonucleotide column. The Kd of E2 binding to a 41-bp oligonucleotide containing a single binding site was found to be 2 x 10(-11) M. When two binding sites were included on an oligonucleotide, cooperative binding to these sites by the E2 protein was observed. A cooperativity parameter of 8.5 was determined for E2 binding to two sites. An 86-amino-acid peptide encompassing the C terminus of the protein retains the ability to bind E2 binding sites with a Kd of 4 x 10(-10) M but exhibits slight cooperativity of binding to two adjacent sites. A major determinant for cooperative binding of the full-length E2 protein is thus encoded by the N-terminal amino acids outside the minimal DNA binding domain.

Baculoviridae↗
PubMed1991

Human papillomavirus DNA sequences in cell lines derived from head and neck squamous cell carcinomas.

There is increasing evidence that human papillomaviruses (HPV) have a casual role in some neoplasms in human beings. As examples, DNA of HPV types 16, 18, and 31 are frequently present in genital cancers in humans. Recently, oncogenic HPV types have also been identified in neoplasms of the head and neck, including verrucous carcinoma of the larynx, squamous cell carcinomas of the oral cavity and larynx, and inverted papillomas of the nose. These findings and our resource of an extensive panel of head and neck squamous cell carcinoma (HNSCC) cell lines led us to begin to investigate how frequently HPV DNA was present in these tumor cell lines. For initial analysis, twenty-two HNSCC cell lines derived from 20 patients' tumors were selected as representative of our tumor cell line panel with respect to diversity of primary site, tumor stage, patient age, sex, and clinical course. For Southern analysis, cell line DNA was tested for hybridization with DNA probes for HPV types 6, 11, 16, 18, and 31. Polymerase chain reaction (PCR) analysis was also performed on five tumor cell lines using types 6, 11, 16, 18, and 52 as probes. Southern blot analysis revealed HPV-specific signals in two of the 22 HNSCC cell lines tested. One of these, UM-SCC-23, was HPV 31 positive, which to our knowledge is the first identification of HPV 31 in HNSCC. UM-SCC-63, the other HPV-positive tumor identified by Southern analysis, hybridized with both type 18 and 31. Of the five tumor cell lines tested with PCR, two were HPV positive.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗