Proteasomal Degradation of p53 by Human Papillomavirus E6 Oncoprotein Relies on the Structural Integrity of p53 Core Domain

The E6 oncoprotein produced by high-risk mucosal HPV stimulates ubiquitinylation and proteasome-dependent degradation of the tumour suppressor p53 via formation of a trimeric complex comprising E6, p53, and E6-AP. p53 is also degraded by its main cellular regulator MDM2. The main binding site of p53 to MDM2 is situated in the natively unfolded N-terminal region of p53. By contrast, the regions of p53 implicated in the degradation by viral E6 are not fully identified to date. Here we generated a series of mutations (Y103G, Y107G, T155A, T155V, T155D, L264A, L265A) targeting the central folded core domain of p53 within a region opposite to its DNA-binding site. We analysed by in vitro and in vivo assays the impact of these mutations on p53 degradation mediated by viral E6 oncoprotein. Whereas all mutants remained susceptible to MDM2-mediated degradation, several of them (Y103G, Y107G, T155D, L265A) became resistant to E6-mediated degradation, confirming previous works that pointed to the core domain as an essential region for the degradation of p53. In parallel, we systematically checked the impact of the mutations on the transactivation activity of p53 as well as on the conformation of p53, analysed by Nuclear Magnetic Resonance (NMR), circular dichroism (CD), and antibody probing. These measurements suggested that the conformational integrity of the core domain is an essential parameter for the degradation of p53 by E6, while it is not essential for the degradation of p53 by MDM2. Thus, the intracellular stability of a protein may or may not rely on its biophysical stability depending on the degradation pathway taken into consideration

Splicing misregulation of SCN5A contributes to cardiac-conduction delay and heart arrhythmia in myotonic dystrophy

Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing changes. Cardiac alterations, characterized by conduction delays and arrhythmia, are the second most common cause of death in DM. Using RNA sequencing, here we identify novel splicing alterations in DM heart samples, including a switch from adult exon 6B towards fetal exon 6A in the cardiac sodium channel, SCN5A. We find that MBNL1 regulates alternative splicing of SCN5A mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability compared with the control adult isoform. Importantly, reproducing splicing alteration of Scn5a in mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant features of myotonic dystrophy. In conclusion, misregulation of the alternative splicing of SCN5A may contribute to a subset of the cardiac dysfunctions observed in myotonic dystrophy.Peer reviewe

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

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

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

Transactivation activity of p53 mutants.

<p>(A) H1299 cells were co-transfected by vectors coding for p53 proteins and a vector expressing the reporter gene luciferase under the control of the p21 promoter (NT: Not Transfected). Crude cellular extracts were then analysed by luminescence dosage (AU: Arbitrary Units). (B) H1299 cells were co-transfected by vectors for transient expression p53-WT and mutant proteins. 24 h after transfection, crude extracts were separated on 12% SDS-PAGE and analysed by Western blotting using monoclonal anti-p53 antibody, anti-p21 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