HPV E6-mediated p53 degradation.

<p>A) HA-tagged p53 levels were visualized by Western blot after co-transfection with HA-tagged HPV E6 from HPV11, HPV16 and HPV18 into C-33A cells. Lanes 1, 3, 5 and 7 show results from co-transfection of HA-p53 and vectors indicated at the top of the figure. Lanes 2, 4, 6 and 8 show the p53 levels after treatment with MG132, as indicated at the top of the figure. β-tubulin was visualized as a loading control. (B) Half life of HA-p53 in transfected 293T cells. 293T cells were transfected with HA-p53 and control (pQCXIN) or E6 ORFs (HPV11, HPV18, HPV53, HPV56 and HPV66). The band intensities were determined from the scanned Western blot using ImageQuant and the signals at time 0 were defined as 100%. The band intensities of the indicated time points were normalized to time 0.</p

Alignment of HPV alpha E6 ORFs.

<p>The alignment of all 27 E6 ORFs tested in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone-0012816-g002" target="_blank">Figure 2</a> is shown. HPV types degrading p53 are shown at the top of the alignment and those not degrading p53 are shown at the bottom. The shaded region represents a proposed E6-AP binding domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Liu1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Pim1" target="_blank">[26]</a>. The amino acid sequences of the E6 ORFs were aligned using Clustal X (version 1.81) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Thompson1" target="_blank">[36]</a>. The amino acid at position 31 (arrow, <b>bold</b>) was associated with p53 degradation (p<0.01). “_” indicate gaps, whereas “.” indicates identical residues with the HPV16 E6 amino acid sequence shown in the top row.</p

Phylogeny of alpha-HPVs and degradation of p53 by HPV E6 ORFs.

<p>HPV E6 activity on HA-p53 steady state levels were determined by Western blot using the assay shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone-0012816-g001" target="_blank">Figure 1A</a>. The phylogenetic tree at the left was constructed using the combined early gene sequences as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Narechania1" target="_blank">[35]</a>. Epidemiological carcinogenicity was extrapolated from recent reviews <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Schiffman1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Bouvard1" target="_blank">[24]</a> and is indicated in the column labeled “carcinogenic risk”: ++, highly oncogenic; +, oncogenic; +/−, probably oncogenic; −, not significantly associated with cervix cancer; NA, insufficient data. The p53 levels after co-transfection with E6 from each type indicated on the left are shown in the far right column labeled, “p53” (with and without MG132) and the results are summarized in the column labeled“↓p53”. Endogenous β-tubulin (far right column) represents a loading control. The alpha-HPV species groups are indicated by brackets with a number to the right. The empty vector control, pQCXIN is shown at the bottom.</p

HPV E6 mutagenesis and degradation of p53.

<p>(A) The steady state levels of HA-p53 co-transfected with wild type or mutant HPV71, 90 and 106 HA-E6 in C-33A cells are shown in the row labeled HA-p53. The unmodified E6 ORFs are indicated with “WT” and the mutated E6 ORFs are indicated with the replacement amino acid at the top of the figure. β-tubulin is shown as a loading control in the bottom row. (B) Representative images of double immuno-fluorescence experiments. HA-tagged HPV E6 constructs are green, whereas the endogenous p53 is labeled red. In the p53 image, arrows indicate the location of the E6 expressing cells. Absence of p53 signal (red) is the result of degradation. In the merged images, yellow signifies a non-degrader, whereas green only (HPV E6) is indicative of a p53 degrader. Cell nuclei are detected with DAPI and are shown in the bottom row. Each HPV E6 construct is indicated at the bottom of each column.</p

Degradation of p53 wild type and indicated mutants via E6.

<p>(A) Radiolabeled p53 (wild type and indicated mutants) and HPV16 E6 proteins produced in rabbit reticulocyte lysates were incubated together at 28°C. Aliquots were removed at the indicated times before separation by 12% SDS-PAGE and exposition to photographic film. (B) Same experiment as in A, but levels of radiolabeled p53, after exposition to photographic film, were quantified by densitometry (BIO-RAD, Quantity One Software). Mean values (AU) ± standard deviation for three independent experiments are shown. (C) H1299 cells were co-transfected by vectors for transient expression of HPV16 E6 and p53 proteins. Where indicated, 4 h prior to harvesting, the medium was supplemented with the 26S proteasome inhibitor ALLN at a final concentration of 100 µM. 24 h after transfection, extracted proteins were separated by 12% SDS-PAGE and analysed by Western-blotting using monoclonal anti-p53 antibody, polyclonal anti-actin antibody and monoclonal anti-16-E6 antibody.</p

Biophysical analysis of Wt and mutant p53 core domain.

<p>(A) Comparison of different p53 core domain proteins (WT, L265A, Y103G) with respect to secondary structure content by CD. The spectra were recorded in 20 mM sodium phosphate (pH 6.8), 20 mM NaCl, 2 mM DTT at 10°C. The far-UV spectrum of the p53wt and p53Y103G are similar and show characteristics of folded proteins with a minimum at 201 nm while the spectrum of the L265A p53 core domain suggests a large proportion of unfolded protein as indicated by the shift of the minimum towards smaller wavelength and a negative signal at 200 nm. (B) Thermal denaturation of the p53 core domain proteins monitored by far-UV CD spectroscopy at 210 nm. The spectra were recorded in 20 mM sodium phosphate (pH 6.8), 50 mM NaCl, 2 mM DTT. For clarity, spectra have been offset by 10 mdeg between each curve. (C) ¹H-¹⁵N correlation spectra of p53 core domain (residues 94–312) acquired at 10°C on a Bruker DRX600 spectrometer equipped with a z-gradient triple resonance cryoprobe. The p53WT core domain is represented in black (left panel), the p53L265A core domain in red (middle panel) and the p53Y103G core domain in blue (right panel).</p

MDM2-mediated degradation of p53 mutants.

<p>H1299 cells were co-transfected by vectors for transient expression of MDM2 and p53 proteins. 24 h after transfection, extracted proteins were separated by 10% SDS-PAGE and analysed by Western blotting using monoclonal anti-p53 antibody, polyclonal anti-actin antibody.</p

Localization of residues within the structure of p53 core domain.

<p>(A) Schematic view of the domain structure of p53. The 393-residue p53 protein comprises an N-terminal transactivation domain (blue), followed by a proline-rich region (purple), a central DNA-binding core domain (green), a tetramerization domain (red) and a regulatory domain (yellow) at the extreme C-terminus. The regions of possible interaction between p53 and MDM2 or p53 and HPV E6 are indicated. (B) Enlarged view of the three-dimensional structure of p53 core domain. Mutants analysed for this study are all localised in the same tridimensional region, distal from the DNA binding site. The leucine 265 is shown in light green, the leucine 264 in dark green, the threonine 155 in orange, the tyrosine 103 in pink, the tyrosine 107 in purple and the region in yellow corresponds to the residues 99 to 107. The β-strands are shown in blue (S7, S9 and S10) and the a-helix in red. The view was created from PDB entry: 1TSR using the PyMOL software.</p

Behaviour of p53 mutants in presence of E6.

<p>(A) Radiolabeled p53 (wild type and indicated mutants) and HPV16 E6 proteins produced in rabbit reticulocyte lysates were incubated together at 28°C. Aliquots were removed at the indicated times before separation by 12% SDS-PAGE and exposition to photographic film. (B, C and D) H1299 cells were co-transfected by vectors for transient expression of HPV16 E6 and p53 proteins. Where indicated, 4 h prior to harvesting, the medium was supplemented with the 26S proteasome inhibitor ALLN at a final concentration of 100 µM. 24 h after transfection, extracted proteins were separated by 12% SDS-PAGE and analysed by Western-blotting using monoclonal anti-p53 antibody, polyclonal anti-actin antibody and monoclonal anti-16-E6 antibody.</p

Conformation of p53 mutants.

<p>H1299 cells were transfected by vectors for transient expression of p53 proteins. Cell lysates were incubated with mouse monoclonal anti-p53 antibody, either the Pab 1620 recognising only the “wild-type” conformation epitope or the Pab 240 recognising only the “mutant” conformation epitope. Immune complexes and whole lysates (input) were separated by 12% SDS-PAGE and subjected to Western blotting using antibodies against p53 (polyclonal rabbit).</p