scholarly journals Membrane Orientation of the Human Papillomavirus Type 16 E5 Oncoprotein

2009 ◽  
Vol 84 (4) ◽  
pp. 1696-1703 ◽  
Author(s):  
Ewa Krawczyk ◽  
Frank A. Suprynowicz ◽  
Sawali R. Sudarshan ◽  
Richard Schlegel
Keyword(s):  
Human Papillomavirus ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Biologically Active ◽  
Human Papillomavirus Type 16 ◽  
Cytoplasmic Proteins ◽  
C Terminus ◽  
Low Concentrations ◽  
E5 Protein ◽  
E5 Oncoprotein

ABSTRACT The E5 protein of human papillomavirus type 16 is a small, hydrophobic protein that localizes predominantly to membranes of the endoplasmic reticulum (ER). To define the orientation of E5 in these membranes, we employed a differential, detergent permeabilization technique that makes use of the ability of low concentrations of digitonin to selectively permeabilize the plasma membrane and saponin to permeabilize all cellular membranes. We then generated a biologically active E5 protein that was epitope tagged at both its N and C termini and determined the accessibility of these termini to antibodies in the presence and absence of detergents. In both COS cells and human ectocervical cells, the C terminus of E5 was exposed to the cytoplasm, whereas the N terminus was restricted to the lumen of the ER. Finally, the deletion of the E5 third transmembrane domain (and terminal hydrophilic amino acids) resulted in a protein with its C terminus in the ER lumen. Taken together, these topology findings are compatible with a model of E5 being a 3-pass transmembrane protein and with studies demonstrating its C terminus interacting with cytoplasmic proteins.

scholarly journals Two transmembrane dimers of the bovine papillomavirus E5 oncoprotein clamp the PDGF β receptor in an active dimeric conformation

2017 ◽  
Vol 114 (35) ◽  
pp. E7262-E7271 ◽  
Author(s):  
Alexander G. Karabadzhak ◽  
Lisa M. Petti ◽  
Francisco N. Barrera ◽  
Anne P. B. Edwards ◽  
Andrés Moya-Rodríguez ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Receptor Activation ◽  
Bovine Papillomavirus ◽  
Detailed Model ◽  
E5 Protein ◽  
Membrane Modeling ◽  
E5 Oncoprotein ◽  
Β Receptor ◽  
Dynamics Simulations

The dimeric 44-residue E5 protein of bovine papillomavirus is the smallest known naturally occurring oncoprotein. This transmembrane protein binds to the transmembrane domain (TMD) of the platelet-derived growth factor β receptor (PDGFβR), causing dimerization and activation of the receptor. Here, we use Rosetta membrane modeling and all-atom molecular dynamics simulations in a membrane environment to develop a chemically detailed model of the E5 protein/PDGFβR complex. In this model, an active dimer of the PDGFβR TMD is sandwiched between two dimers of the E5 protein. Biochemical experiments showed that the major PDGFβR TMD complex in mouse cells contains two E5 dimers and that binding the PDGFβR TMD to the E5 protein is necessary and sufficient to recruit both E5 dimers into the complex. These results demonstrate how E5 binding induces receptor dimerization and define a molecular mechanism of receptor activation based on specific interactions between TMDs.

scholarly journals The Human Papillomavirus Type 16 E5 Oncoprotein Inhibits Epidermal Growth Factor Trafficking Independently of Endosome Acidification

2010 ◽  
Vol 84 (20) ◽  
pp. 10619-10629 ◽  
Author(s):  
Frank A. Suprynowicz ◽  
Ewa Krawczyk ◽  
Jess D. Hebert ◽  
Sawali R. Sudarshan ◽  
Vera Simic ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Surface ◽  
Vesicle Fusion ◽  
Ph Sensitive ◽  
Biologically Active ◽  
Human Papillomavirus Type 16 ◽  
E5 Oncoprotein ◽  
Epidermal Growth

ABSTRACT The human papillomavirus type 16 E5 oncoprotein (16E5) enhances acute, ligand-dependent activation of the epidermal growth factor receptor (EGFR) and concomitantly alkalinizes endosomes, presumably by binding to the 16-kDa “c” subunit of the V-ATPase proton pump (16K) and inhibiting V-ATPase function. However, the relationship between 16K binding, endosome alkalinization, and altered EGFR signaling remains unclear. Using an antibody that we generated against 16K, we found that 16E5 associated with only a small fraction of endogenous 16K in keratinocytes, suggesting that it was unlikely that E5 could significantly affect V-ATPase function by direct inhibition. Nevertheless, E5 inhibited the acidification of endosomes, as determined by a new assay using a biologically active, pH-sensitive fluorescent EGF conjugate. Since we also found that 16E5 did not alter cell surface EGF binding, the number of EGFRs on the cell surface, or the endocytosis of prebound EGF, we postulated that it might be blocking the fusion of early endosomes with acidified vesicles. Our studies with pH-sensitive and -insensitive fluorescent EGF conjugates and fluorescent dextran confirmed that E5 prevented endosome maturation (acidification and enlargement) by inhibiting endosome fusion. The E5-dependent defect in vesicle fusion was not due to detectable disruption of actin, tubulin, vimentin, or cytokeratin filaments, suggesting that membrane fusion was being directly affected rather than vesicle transport. Perhaps most importantly, while bafilomycin A1 (like E5) binds to 16K and inhibits endosome acidification, it did not mimic the ability of E5 to inhibit endosome enlargement or the trafficking of EGF. Thus, 16E5 alters EGF endocytic trafficking via a pH-independent inhibition of vesicle fusion.

scholarly journals Structural and functional aspects of the multiplicity of Neu differentiation factors

1994 ◽  
Vol 14 (3) ◽  
pp. 1909-1919
Author(s):  
D Wen ◽  
S V Suggs ◽  
D Karunagaran ◽  
N Liu ◽  
R L Cupples ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Transmembrane Domain ◽  
Structural Heterogeneity ◽  
Binding Activity ◽  
Biologically Active ◽  
Specific Rate ◽  
C Terminus ◽  
Functional Aspects ◽  
Differentiation Factors ◽  
Variable Domains

We used molecular cloning and functional analyses to extend the family of Neu differentiation factors (NDFs) and to explore the biochemical activity of different NDF isoforms. Exhaustive cloning revealed the existence of six distinct fibroblastic pro-NDFs, whose basic transmembrane structure includes an immunoglobulin-like motif and an epidermal growth factor (EGF)-like domain. Structural variation is confined to three domains: the C-terminal portion of the EGF-like domain (isoforms alpha and beta), the adjacent juxtamembrane stretch (isoforms 1 to 4), and the variable-length cytoplasmic domain (isoforms a, b, and c). Only certain combinations of the variable domains exist, and they display partial tissue specificity in their expression: pro-NDF-alpha 2 is the predominant form in mesenchymal cells, whereas pro-NDF-beta 1 is the major neuronal isoform. Only the transmembrane isoforms were glycosylated and secreted as biologically active 44-kDa glycoproteins, implying that the transmembrane domain functions as an internal signal peptide. Extensive glycosylation precedes proteolytic cleavage of pro-NDF but has no effect on receptor binding. By contrast, the EGF-like domain fully retains receptor binding activity when expressed separately, but its beta-type C terminus displays higher affinity than alpha-type NDFs. Likewise, structural heterogeneity of the cytoplasmic tails may determine isoform-specific rate of pro-NDF processing. Taken together, these results suggest that different NDF isoforms are generated by alternative splicing and perform distinct tissue-specific functions.

Cellular proteins associated with the E5 oncoprotein of human papillomavirus type 16

1994 ◽  
Vol 22 (3) ◽  
pp. 333S-333S ◽  
Author(s):  
BARBARA KELL ◽  
RICHARD J JEWERS ◽  
JOHN CASON ◽  
JENNIFER M BEST
Keyword(s):  
Human Papillomavirus ◽  
Cellular Proteins ◽  
Human Papillomavirus Type 16 ◽  
E5 Oncoprotein

scholarly journals E5 protein of human papillomavirus type 16 selectively downregulates surface HLA class I

2004 ◽  
Vol 113 (2) ◽  
pp. 276-283 ◽  
Author(s):  
G. Hossein Ashrafi ◽  
Mohammad R. Haghshenas ◽  
Barbara Marchetti ◽  
Philippa M. O'Brien ◽  
M. Saveria Campo
Keyword(s):  
Human Papillomavirus ◽  
Hla Class I ◽  
Class I ◽  
Human Papillomavirus Type 16 ◽  
E5 Protein

scholarly journals Cytotoxic T-Lymphocyte Responses to Human Papillomavirus Type 16 E5 and E7 Proteins and HLA-A*0201-Restricted T-Cell Peptides in Cervical Cancer Patients

2007 ◽  
Vol 81 (6) ◽  
pp. 2869-2879 ◽  
Author(s):  
Dai-Wei Liu ◽  
Yuh-Cheng Yang ◽  
Ho-Fan Lin ◽  
Mei-Fang Lin ◽  
Ya-Wen Cheng ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Human Papillomavirus ◽  
T Cell ◽  
Cancer Patients ◽  
T Lymphocyte ◽  
Cytotoxic T Lymphocyte ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16 ◽  
E5 Protein ◽  
E7 Proteins

ABSTRACT Previously, we found that human papillomavirus type 16 (HPV-16) E5 protein is a tumor rejection antigen and can induce cytotoxic T-lymphocyte (CTL) activity. Therefore, in this study, human leukocyte antigen A*0201 (HLA-A*0201)-restricted human CTL epitopes of HPV-16 E5 protein were identified using a bioinformatics approach, and the abilities of these predicted peptides to induce an immune response in HLA-A*0201 transgenic mice were confirmed by assaying E5-specific CTLs and in vitro-generated CTLs from normal peripheral blood T lymphocytes of HLA-A2-positive human donors. Second, the CTL responses to HLA-A*0201 CTL epitopes (E5 63-71 and E7 11-20) were examined in HPV-16-infected patients with HLA-A2. Third, the effect of HLA-A-type alleles on CTL activities in response to the entire E5 and E7 proteins was examined in cervical cancer patients. E5 and E7 peptides (but not the whole proteins) stimulated E5- and E7-specific CTL recall responses in HPV-16- and HLA-A2-positive cervical cancer patients, and HPV-16 E5 and E7 proteins stimulated naïve T cells in HPV-16-negative cervical cancer patients with HLA-A11 and -A24 haplotypes. In summary, this is the first demonstration that E5 63-71 is an HLA-A*0201-restricted T-cell epitope of HPV-16 E5.

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