A ubiquitin variant-based affinity approach selectively identifies substrates of the ubiquitin ligase E6AP in complex with HPV-11 E6 or HPV-16 E6

Felix A Ebner,Carolin Sailer,Daniela Eichbichler,Jasmin Jansen,Anna Sladewska-Marquardt,Florian Stengel,Martin Scheffner

doi:10.1074/jbc.ra120.015603

Abstract

The E6 protein of both mucosal high-risk human papillomaviruses (HPVs) such as HPV-16, which have been causally associated with malignant tumors, and low-risk HPVs such as HPV-11, which cause the development of benign tumors, interacts with the cellular E3 ubiquitin ligase E6-associated protein (E6AP). This indicates that both HPV types employ E6AP to organize the cellular proteome to viral needs. However, whereas several substrate proteins of the high-risk E6-E6AP complex are known, e.g. the tumor suppressor p53, potential substrates of the low-risk E6-E6AP complex remain largely elusive. Here, we report on an affinity-based enrichment approach that enables the targeted identification of potential substrate proteins of the different E6-E6AP complexes by a combination of E3-selective ubiquitination in whole-cell extracts and high-resolution MS. The basis for the selectivity of this approach is the use of a ubiquitin variant that is efficiently used by the E6-E6AP complexes for ubiquitination but not by E6AP alone. By this approach, we identified ∼190 potential substrate proteins for low-risk HPV-11 E6 and high-risk HPV-16 E6. Moreover, subsequent validation experiments in vitro and within cells with selected substrate proteins demonstrate the potential of our approach. In conclusion, our data represent a reliable repository for potential substrates of the HPV-16 and HPV-11 E6 proteins in complex with E6AP.

Highlights

Because the coding capacity of viral genomes is rather limited, it is essential for viruses to reprogram the host cell metabolism according to viral need
Whereas infection with low-risk Human papillomaviruses (HPVs) (e.g. HPV-6 and HPV-11) induces the formation of benign lesions with no or only little risk to progress to cancer, women infected with high-risk HPVs (e.g. HPV-16 and HPV18) have a significantly increased risk to develop malignant lesions, most notably cervical cancer [5, 6]
Ubiquitin needs to be equipped with an affinity tag that does not interfere with E6-E6-associated protein (E6AP)–mediated ubiquitination and allows enrichment of ubiquitinated proteins under harsh conditions to minimize the possibility ofpurifying nonubiquitinated proteins

Summary

Introduction

Because the coding capacity of viral genomes is rather limited, it is essential for viruses to reprogram the host cell metabolism according to viral need. To identify potential substrate proteins of both low-risk and high-risk E6 proteins, we established an affinity-based enrichment approach using whole-cell extracts and a dedicated biotin-tagged ubiquitin variant that in the absence of the E6 proteins is only poorly used by E6AP for ubiquitination.

Results

Conclusion