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Biomedical subjects

R Schlegel

Publications and source records attributed to R Schlegel.

At least 19 recordsLinked to original sources
PubMed1992

The BPV-1 E5 protein, the 16 kDa membrane pore-forming protein and the PDGF receptor exist in a complex that is dependent on hydrophobic transmembrane interactions.

The E5 oncoprotein of bovine papillomavirus type 1 is a 44 amino acid, highly hydrophobic protein that induces the stable transformation of immortalized murine fibroblasts, presumably through its activation of growth factor receptors. Previous studies have shown that the E5 protein complexes with the 16 kDa (16k) pore-forming protein of vacuolar H(+)-ATPases. This integral membrane protein is essential for the acidification and function of subcellular compartments that process growth factor receptors. Using an SV40 expression system in COS cells, we analyzed whether the E5-16k complexes bind additional cellular proteins, including growth factor receptors. These studies demonstrate that E5 binds to both the 16k protein and the PDGF receptor and that this tri-component complex can be isolated with antibodies specific for each protein. Importantly, the 16k protein bound to the PDGF receptor in the absence of E5, suggesting that E5 binds to the PDGF receptor via its interaction with the 16k protein. An E5 mutant lacking the hydrophilic carboxyl-terminal 14 amino acids retained binding to both 16k and the PDGF receptor, indicating that E5 binds to these proteins through its hydrophobic, membrane-associating domain. These studies reveal that hydrophobic, intramembrane interactions govern the association of E5, 16k and the PDGF receptor, suggesting a ligand-independent mechanism for receptor activation and a potential link between receptor signal transduction pathways and membrane pore activity.

Adenosine Triphosphatases↗
PubMed1992

Differential effect of tumor necrosis factor on proliferation of primary human keratinocytes and cell lines containing human papillomavirus types 16 and 18.

Keratinocytes immortalized by human papillomaviruses (HPV) 16 and 18 are partially resistant to the inhibition of proliferation exerted by transforming growth factor-beta (TGF-beta). To determine if this finding reflects a generalized resistance to inhibitory cytokines, we studied the effect of tumor necrosis factor-alpha (TNF-alpha) on subconfluent cultures of both normal and HPV-immortalized human foreskin keratinocytes. Whereas primary and HPV-16-immortalized keratinocytes were sensitive to TNF-alpha, HPV-18-immortalized keratinocytes (and those immortalized by simian virus 40) were resistant to the inhibitory effects of this cytokine. The ability of HPV-18 to induce a more resistant phenotype correlated with its more potent in vitro transforming activity and its apparent association with more aggressive tumors. Interestingly, the state of TNF-induced growth inhibition in normal or HPV-16-immortalized keratinocytes was not accompanied by a reduction in the expression of c-myc RNA or protein. This contrasts sharply with the ability of TGF-beta to inhibit c-myc RNA expression in normal cells. Evidently, the resistance of HPV-immortalized keratinocytes to TNF-alpha and TGF-beta proceeds along different regulatory pathways.

Blotting, Northern↗
PubMed1992

The BPV-1 E5 oncoprotein expressed in Schizosaccharomyces pombe exhibits normal biochemical properties and binds to the endogenous 16-kDa component of the vacuolar proton-ATPase.

The 44-amino-acid E5 oncoprotein of bovine papillomavirus type 1 transforms immortalized murine fibroblast cell lines. This highly hydrophobic protein forms homodimers, localizes to intracellular membrane compartments (including the Golgi apparatus), and forms a complex with the 16-kDa membrane-embedded constituent (16k) of the vacuolar proton-ATPase. To develop a system for the genetic and biochemical analysis of the E5/16k interaction, the E5 gene was cloned into a new vector which was designed for expression in the fission yeast Schizosaccharomyces pombe. The E5 protein synthesized in this system dimerized normally and bound to endogenous and overexpressed S. pombe 16k protein. Comparison of the S. pombe and mammalian 16k proteins showed strong conservation in carboxyl-terminal amino acids but greater variation in the amino-terminal sequences, suggesting that E5 was interacting with the 16k carboxyl domains. Finally, a new protein epitope tag is described which permitted for the first time the coprecipitation of E5 with antibodies directed against the 16k protein.

Amino Acid Sequence↗
PubMed1992

HPV-1 L1 protein expressed in cos cells displays conformational epitopes found on intact virions.

Seven polyclonal and monoclonal antibodies were characterized for their ability to react specifically with either conformational or nonconformational epitopes of the HPV-1 virion. Using these antibodies, it was shown that the HPV-1 L1 protein (when expressed by an SV40 vector in cos cells) displayed conformational epitopes characteristic of intact viral particles. In addition, the L1 capsid protein was translocated normally into cell nuclei, was of appropriate size (57 kDa), and could be isolated in native form by immunoprecipitation techniques. Most importantly, the screening of expressed papillomavirus capsid proteins for reactivity with conformation-dependent antibodies represents a new, general methodology for ensuring that such proteins will be suitable for use in vaccine development or in the serologic detection/typing of human papillomavirus infections.

Animals↗
PubMed1992

Mitotic checkpoints.

Entry into mitosis is triggered by activation of maturation promoting factor and a complex of p34cdc2 kinase and cyclin B. Activation induces nuclear lamina breakdown, chromosome condensation and mitotic spindle assembly. Exit from mitosis is initiated by the degradation of cyclin B and the subsequent inactivation of maturation-promoting factor. A more thorough understanding of the checkpoints for initiation of and exit from mitosis has evolved during the past few years.

Animals↗
PubMed1992

A glutamine residue in the membrane-associating domain of the bovine papillomavirus type 1 E5 oncoprotein mediates its binding to a transmembrane component of the vacuolar H(+)-ATPase.

The 44-amino-acid E5 oncoprotein is the major transforming protein of bovine papillomavirus type 1. It is a highly hydrophobic polypeptide which dimerizes and localizes to the Golgi apparatus and endoplasmic reticulum membranes. Recent evidence suggests that E5 modulates the phosphorylation and internalization of the epidermal growth factor and colony-stimulating factor 1 receptors and constitutively activates platelet-derived growth factor receptors in C127 and FR3T3 cells. Although no direct interaction with these growth factor receptors has yet been identified, the E5 oncoprotein has been shown recently to interact with the hydrophobic 16-kDa component of the vacuolar H(+)-ATPase (16K protein) [D. J. Goldstein, M. E. Finbow, T. Andresson, P. McLean, K. Smith, V. Bubb, and R. Schlegel, Nature (London) 352:347-349, 1991]. In the current study, we have further analyzed the E5-16K protein complex by fast protein liquid chromatography and shown that each E5 dimer appears to bind two 16K proteins. In order to define the specific amino acid residues of E5 which participate in this binding, mutated E5 epitope fusion proteins were analyzed for their ability to coprecipitate 16K protein. Transformation-defective mutants containing amino acid substitutions within the short hydrophilic carboxyl-terminal domain retained the ability to associate with the 16K protein. However, E5 mutants lacking the glutamine residue in the hydrophobic domain were markedly inhibited in 16K protein binding. Most interestingly, the placement of a glutamine in several random hydrophobic sequences facilitated 16K protein binding, defining this residue as a potential binding site for the 16K protein component of the proton pump and exemplifying the critical role of hydrophilic amino acids for mediating specific interactions between transmembrane proteins.

Amino Acid Sequence↗
PubMed1992

On the protonophoric activity of oxazolomycin.

By the use of an artificial lipid membrane model we have shown that the antibiotic oxazolomycin (Streptomyces albus) is an effective protonophore at pH < 7.0 but conveys both protons and monovalent cations such as potassium at pH > 7.5 as a passive carrier. The ionophoric properties are suggested to be correlated with its antibacterial, antiviral, and cytotoxic activities.

Electrolytes↗
PubMed1992

Staurosporine overrides checkpoints for mitotic onset in BHK cells.

Under normal conditions, mammalian cells will not initiate mitosis in the presence of either unreplicated or damaged DNA. We report here that staurosporine, a tumor promoter and potent protein kinase inhibitor, can uncouple mitosis from the completion of DNA replication and override DNA damage-induced G2 delay. Syrian hamster (BHK) fibroblasts that were arrested in S phase underwent premature mitosis at concentrations as low as 1 ng/ml, with maximum activity seen at 50 ng/ml. Histone H1 kinase activity was increased to approximately one-half the level found in normal mitotic cells. Inhibition of protein synthesis during staurosporine treatment blocked premature mitosis and suppressed the increase in histone H1 kinase activity. In asynchronously growing cells, staurosporine transiently increased the mitotic index and histone H1 kinase activity but did not induce S phase cells to undergo premature mitosis, indicating a requirement for S phase arrest. Staurosporine also bypassed the cell cycle checkpoint that prevents the onset of mitosis in the presence of damaged DNA. The delay in mitotic onset resulting from gamma radiation was reduced when irradiation was followed immediately by exposure to 50 ng/ml of staurosporine. These findings indicate that inhibition of protein phosphorylation by staurosporine can override two important checkpoints for the initiation of mitosis in BHK cells.

Alkaloids↗
PubMed1991

Chemically induced premature mitosis: differential response in rodent and human cells and the relationship to cyclin B synthesis and p34cdc2/cyclin B complex formation.

Normal eukaryotic cells do not initiate mitosis until DNA replication has been completed. This requirement can be bypassed by exposing cells to certain chemicals. We report here that chemically induced premature mitosis is not readily achieved in all mammalian species. Although hamster cells underwent premature mitosis following treatment with caffeine, the protein phosphatase inhibitor okadaic acid, and the protein kinase inhibitors 2-aminopurine and 6-dimethyl-aminopurine, the mouse and human cells examined in this study displayed little or no response to any of these compounds. Differences in cell permeability or metabolism could not account for the species specificity of these drugs, because other biochemical and mitosis-promoting activities were apparent in human cells. Cell-type specificity can be explained, however, by the timing of cyclin B synthesis and p34cdc2/cyclin B complex formation during the cell cycle. Synthesis of cyclin B and formation of a p34cdc2/cyclin B complex, both of which are required for initiation of mitosis, were prevalent in hamster cells arrested in S phase but were absent or barely detectable in arrested human cells. In hamster cells, the hyperphosphorylated form of p34cdc2 was complexed with cyclin B and underwent tyrosine dephosphorylation during caffeine-induced premature mitosis. These findings indicate that the onset of mitosis is regulated somewhat differently among mammalian cell types and that these differences affect the vulnerability of cells to drug-induced mitotic aberrations and cytogenetic damage.

2-Aminopurine↗
PubMed1991

Bovine papillomavirus E5 oncoprotein binds to the 16K component of vacuolar H(+)-ATPases.

The major transforming protein of bovine papillomavirus type 1, E5, is mainly associated with endomembranes, specifically binding to a cellular protein of relative molecular mass 16,000 (16K). At the same time as transformation, E5 causes the phosphorylation of tyrosine residues in epidermal and platelet-derived growth factor receptors. We show here that the 16K protein associated with E5 is the 16K component of vacuolar ATPases. This protein is known to be an integral membrane protein in endosomes, bovine chromaffin granules, synaptic vesicles, fungal and plant vacuoles and clathrin-coated vesicles, as well as a component of gap-junction-like membrane complexes. Because proton pumps are critical for the function of cellular compartments that process growth-factor receptors, the interaction of E5 with the 16K protein could explain the pleiomorphic features of cells transformed by E5.

Adenosine Triphosphatases↗
PubMed1991

A genomic sequence of the Schizosaccharomyces pombe 16 kDa vacuolar H(+)-ATPase.

We have isolated the gene encoding the 16 kDa vacuolar H(+)-ATPase from Schizosaccharomyces pombe. On the basis of RNA splicing signals and amino acid sequence homology with other 16 kDa H(+)-ATPases, the genomic DNA sequence indicated the 16 kDa protein is encoded by five exons. The C-terminal 50 amino acids has more than 90% homology with vacuolar H(+)-ATPases of mammalian cells.

Amino Acid Sequence↗
PubMed1991

Differences in transformation activity between HPV-18 and HPV-16 map to the viral LCR-E6-E7 region.

Homologous, subgenomic fragments of the viral LCR and E6/E7 transforming genes of HPV-18 and HPV-16 were amplified from several primary cervical, penile, and vulvar tumors and cloned into a pUC-18-derived vector. When assayed by a quantitative transformation assay using primary human keratinocytes, the subgenomic regions of HPV-16 and HPV-18 exhibited transforming activities similar to that of the full-length, prototype HPV genomes. More importantly, the HPV-18 LCR-E6-E7 region was approximately 10- to 50-fold more active than that of HPV-16. These studies demonstrate (1) that the transforming activity differences previously observed between prototype HPV-16 and HPV-18 map to the LCR-E6-E7 region, and (2) that individual and independent isolates of HPV-16 and HPV-18 exhibit consistent differences in transforming potential, even when isolated from different anatomic sites.

Base Sequence↗
PubMed1991

The viral transcriptional regulatory region upstream of the E6 and E7 genes is a major determinant of the differential immortalization activities of human papillomavirus types 16 and 18.

The human papillomavirus types 16 (HPV-16) and 18 (HPV-18) can immortalize primary human keratinocytes. The region of the viral genome responsible for this function maps to the E6 and E7 genes and their respective upstream transcriptional regulatory sequences, the long control regions (LCRs). The HPV-18 LCR/E6/E7 is more efficient in this immortalization function than the analogous region of the HPV-16 genome, resembling the difference in the immortalization potentials of the two full-length viral genomes. This study was designed to examine the basis for the difference in HPV-16 and HPV-18 immortalization efficiencies. The E6 and E7 genes of either HPV-16 or HPV-18, when expressed from the same heterologous promoter, immortalized primary human keratinocytes with the same low efficiency, suggesting that the difference in immortalization activities was not due to the different E6 or E7 genes themselves but rather to a difference in the transcriptional regulatory regions upstream of these genes. The analysis of a series of chimeric HPV-16 and HPV-18 LCR/E6/E7 constructs confirmed this observation and further mapped the viral element responsible for the major difference in immortalization efficiency to the transcriptional regulatory region upstream of the E6 and E7 genes.

Base Sequence↗
PubMed1991

In vitro photodynamic treatment of normal and human papilloma virus-transfected keratinocytes with photofrin II and red light.

Photodynamic therapy involves the use of light of appropriate wavelength to excite a photosensitizer to result in tissue destruction. The photosensitizer dihematoporphyrin ether and red light were used to treat normal and human papilloma virus type 18-transfected keratinocytes in vitro. Although both cell lines were sensitive to treatment, normal keratinocytes retained more dihematoporphyrin ether and were more sensitive to photodynamic therapy than were transfected cells. In vitro data fail to show the selective retention of dihematoporphyrin ether reported elsewhere in the literature in papillomas and tumors. We did not find selective uptake or retention of dihematoporphyrin ether by human papilloma virus-transfected cells, despite previously published in vivo data to the contrary. Dihematoporphyrin ether retention and thus selective photosensitivity of papillomas in vivo is not caused by individual cellular differences in the processing of dihematoporphyrin ether. In addition, because in vitro results may not parallel in vivo results, both in vivo and in vitro models must be investigated in the study of phototherapeutic agents.

Antiviral Agents↗
PubMed1990

TGF-beta 1 inhibition of c-myc transcription and growth in keratinocytes is abrogated by viral transforming proteins with pRB binding domains.

TGF-beta 1 is demonstrated to inhibit skin keratinocyte proliferation when added during the G1 phase of the cell cycle. Human foreskin keratinocytes transformed with either HPV-16 or -18 or SV40, however, were resistant to the growth inhibitory effects of TGF-beta 1. Since TGF-beta 1 appears to inhibit keratinocyte growth through down-regulation of c-myc, it was hypothesized that these DNA tumor viruses might be modulating the response to TGF-beta 1 via this pathway. Transient expression of proteins HPV-16 E7, adenovirus type 5 E1A, and SV40 large T antigen is demonstrated to block TGF-beta 1 suppression of c-myc transcription. This effect was not observed with DNA tumor virus transforming proteins mutated in their pRB binding domain. These observations indicate that pRB or another protein that interacts with this binding domain mediates TGF-beta 1 regulation of c-myc gene expression and growth inhibition.

Adenovirus Early Proteins↗
PubMed1990

The E5 oncoprotein of bovine papillomavirus binds to a 16 kd cellular protein.

The E5 oncoprotein of bovine papillomavirus type 1 is the smallest known viral transforming protein. It is a 44 amino acid polypeptide asymmetrically oriented in Golgi and plasma membranes which appears to modify (either directly or indirectly) the internalization and phosphorylation of at least two growth factor receptors: EGF and CSF-1. To identify cellular proteins associated with E5, we have constructed two E5 fusion proteins, each of which contains a well-characterized epitope at the E5 amino terminus. These E5-epitope fusion proteins are biologically active, localize normally to cellular membranes and form dimers. Both monoclonal and polyclonal antibodies against the inserted epitopes specifically co-precipitate E5 and an associated 16 kd cellular protein. A transformation-defective E5 mutant containing a substitution within the hydrophobic portion of E5 is defective in its ability to bind the 16 kd protein. These findings suggest a role for E5/16 kd binding in the process of cellular transformation.

Animals↗