Abstract
Cervical cancer is caused by a persistent infection of the mucosal epithelia with high-risk human papilloma viruses (HPVs). The viral oncoprotein E6 is responsible for the inactivation of the tumour suppressor p53 and thus plays a crucial role in HPV-induced tumorigenesis. The viral E6 protein forms a trimeric complex with the endogenous E3 ubiquitine ligase E6AP and the DNA-binding domain (DBD) of p53, which results in the polyubiquitination and proteasomal degradation of p53. We have developed nanobodies (Nbs) against the DBD of p53, which substantially stabilise p53 in HeLa cells. The observed effect is specific for HPV-infected cells, since similar effects were not seen for U2OS cells. Despite the fact that the stabilised p53 was strongly nuclear enriched, its tumour suppressive functions were hampered. We argue that the absence of a tumour suppressive effect is caused by inhibition of p53 transactivation in both HPV-infected and HPV-negative cells. The inactivation of the transcriptional activity of p53 was associated with an increased cellular proliferation and viability of HeLa cells. In conclusion, we demonstrate that p53 DBD Nbs positively affect protein stability whilst adversely affecting protein function, attesting to their ability to modulate protein properties in a very subtle manner.
Highlights
Cervical cancer is the fourth most common cancer in woman worldwide with a ratio of mortality to incidence of 52%
There are 12 characterized high-risk human papilloma viruses (HPVs), of which HPV16 and HPV18 are responsible for 71% of all cervical cancers[3,20]
The viral E6 protein targets p53 for excessive proteasomal degradation which renders the tumour suppressor unable to induce apoptosis in response to the abnormal cellular proliferation caused by E7-mediated inhibition of pRb
Summary
Cervical cancer is the fourth most common cancer in woman worldwide with a ratio of mortality to incidence of 52%. The viral, non-enzymatic, proteins E6 and E7 are fundamental for HPV-induced carcinogenesis Both proteins interfere with pathways involved in cellular transformation or the immune response through the formation of complexes with cellular proteins. This would trigger the activation of p53 and would result in the elimination of the cell via cell cycle arrest or apoptosis This response is blocked since E6 targets p53 for proteasomal degradation via the formation of a trimeric p53-E6/E6AP complex[7,8]. Previous research has demonstrated the potency of single-domain antibodies, known as Nbs, to block interactions between proteins[10] In this regard, we aimed to target the p53-E6/E6AP interaction by means of Nbs that were developed against the DBD of p53. Given the impact of the p53 DBD Nbs on p53 functionality, it is of interest to identify the targeted epitopes in order to unravel the mechanisms behind the inactivation of p53
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