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Vats, Arushi
(2021).
DOI: https://doi.org/10.21954/ou.ro.00013cd6
Abstract
The high-risk Human Papillomavirus (HPV) E6 oncoprotein is known to contribute to human malignancy by targeting several of its cellular substrates for degradation through the ubiquitin-proteasome pathway. Whilst E6AP plays a critical role in targeting p53, its role in the degradation of many other E6 substrates is unclear because loss of E6AP also induces E6 degradation. To examine this further, we used CRISPR-edited E6AP knockout (E6AP K/O) cells to perform E6 degradation assays in the presence of a catalytically inactive mutant form of E6AP, thus ensuring the stabilization of E6, but with the ligase itself being functionally inactive. We found that E6 can mediate the degradation of several PDZ domain-containing proteins independently of E6AP ubiquitin ligase activity. Using this system, we also aimed to identify the ligases potentially responsible for degrading E6 in the absence of E6AP by performing a high-throughput human siRNA library screen against ubiquitin ligases, using expression of GFP-tagged HPV-18E6 as the reporter. We found a number of ubiquitin ligases whose knockdown rescued E6 protein levels, with FBXO4 being the best candidate. We also found that loss of E6AP also induces a dramatic increase in the levels of phosphorylated E6 (pE6), despite the expected overall reduction in total E6 protein levels. Phosphorylation of E6 requires transcriptionally active p53 and occurs in a manner that is dependent upon DNA PK. These results identify a novel feedback loop, where loss of E6AP results in upregulation of p53, leading to increased levels of E6 phosphorylation, which, in turn. correlates with increased association with 14-3-3 and inhibition of p53 transcriptional activity. Finally, we show that knockdown of E6AP in HPV-positive cervical cancer-derived cells causes a marked decrease in E7 protein levels. This is due to a decrease in the E7 half-life and occurs in a proteasome-dependent manner. In an attempt to define the underlying mechanism, we show that E7 can also associate with E6AP, albeit in a manner different from that of E6. In addition, we show that E6AP-dependent stabilization of E7 also leads to an increase in the degradation of E7’s cellular target substrates. Interestingly, ectopic over-expression of E6 protein results in lower levels of E7 protein, through E6's sequestration of E6AP, demonstrating a surprising interplay between E6 and E7, in manner that is mediated by the E6AP ubiquitin ligase.
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CORE (COnnecting REpositories)
