Researchers and clinicians are fairly certain that all cervical cancers are caused by Human Papillomavirus (HPV) infections, and that HPV16 and HPV18 are responsible for about 70% of all cases. HPV16 and HPV18 have also been shown to cause almost half the vaginal, vulvar, and penile cancers, while about 85% of anal cancers are also caused by HPV16.
E6 is a potent oncogene of HR-HPVs, and its role in progression to malignancy continues to be explored. The E6 oncoprotein of HPV can promote viral DNA replication through several pathways. It forms a complex with human E3-ubiquitin ligase E6-associated protein (E6AP), which can in turn target the p53 tumor-suppressor protein, leading to its ubiquitin-mediated degradation. In particular, E6 from HR-HPVs can block apoptosis, activate telomerase, disrupt cell adhesion, polarity and epithelial differentiation, alter transcription and G-protein signaling, and reduce immune recognition of HPV-infected cells.
In a recent publication a new procedure generated a stable, unmutated HPV16 E6 protein (1). The His6-tagged E6 proteins from HPV16, HPV18, and HPV11 E6 genes without any further modification in the amino-acid sequence were produced in bacteria as soluble and stable molecules. Structural analyses of HPV16 His6-E6 suggested that it maintains correct folding and conformational properties. C57BL/6 mice immunized with HPV16 His6-E6 developed significant humoral immune responses. The E6 proteins from HPV16, HPV18, and HPV11 were purified according to a new procedure, and investigated for protein–protein interactions. HR-HPV His6-E6 bound p53, the PDZ1 motif from MAGI-1 proteins, the human discs large tumor suppressor, and the human ubiquitin ligase E6-associated protein, thus suggesting that it is biologically active.
The purified HR-HPV E6 proteins were also characterized to determine if they targeted the MAGI-3 and p53 proteins for degradation. Details for application are as follows: The DNA of pSP64-HPV16 E6, pSP64-p53-pro and pCDNA3-MAGI-3 plasmids was transcribed and translated in vitro in a rabbit reticulocyte lysate (TNT System with radiolabeling with 0.6 mCi/mL 35[S]-cysteine. The translation efficiency was monitored by analyzing 1µL aliquots of each protein using SDS-PAGE and PhosphorImager analysis.
In the standard in vitro degradation assay degradation was monitored by mixing the translated target proteins with 50 ng of the purified His6-E6 protein at a 3:1 ratio with an incubation at 25 °C. After 1 h and 2 h, 5-µL aliquots were removed from the reaction mixtures and analyzed. The samples were added to 5X loading buffer boiled and analyzed using SDS-PAGE and autoradiography.
The degradation experiments were carried out three times, and the relative quantification was performed with a PhosphorImager by measuring the signal intensities of protein bands.
When MAGI-3 was combined with HPV16 or HPV18 His6-E6, a very weak band for MAGI-3 was seen at T1 that had almost disappeared at T2. In contrast, this band was seen at both T1 and T2 for HPV11 His6-E6. Similar results were observed with p53, where a very weak p53 band was seen at T1 for all three HPV His6-E6 proteins, but only for HPV11 His6-E6 at T2. As controls, MAGI-3 and p53 proteins were incubated in the absence of His6-E6 and the in vitro translated HPV16 E6 protein alone was used in the incubations.
Illiano, E. et al. (2016) Production of functional, stable, unmutated recombinant human papillomavirus E6 oncoprotein: implications for HPV-tumor diagnosis and therapy. J. Transl. Med. 14, 224–38.
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