E6AP is a HECT type E3 ubiquitin (UB) ligase that is associated with the E6 protein of human papillomavirus (HPV) to induce the ubiquitination and degradation of tumor suppressor protein p53 and promote the development of cervical cancer. We recently developed an orthogonal ubiquitin transfer (OUT) cascade to profile the substrate of E6AP in HEK293 cells. To enable OUT, we engineered UB to generate xUB to enable its exclusive transfer through an engineered UB transfer cascade consisting of xE1, xE2 and xE3 enzymes to the substrates of a specific E3. We expressed affinity-tagged xUB and the xE1-xE2-xE3 cascade in the cell, affinity-purified xUB-conjugated proteins, and identified them by proteomics to profile the E3 substrates. Here, we used OUT to generate the substrate profiles of E6AP with and without E6 expression in HEK293 cells to reveal how HPV E6 redirects the ubiquitination targets of E6AP. We found E6 promotes the ubiquitination and degradation of importins by E6AP. Since importins are responsible for transporting transcription factors such as STATs into the nucleus to activate an antiviral response, the E6 stirred targeting of importin by E6AP may suppress host cell defense against HPV infection. We have also developed OUT cascades with RBR E3s Traid3a that is important for neurodevelopment.

Stirring of the ubiquitination targets of E6AP by human papillomavirus protein E6 revealed by orthogonal ubiquitin transfer (OUT)

ACS Fall 2021

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