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Abstract The E6 protein of the human papillomavirus (HPV) underpins important protein interaction networks between the virus and host to promote viral infection. Through its interaction with E6AP, a host E3 ubiquitin (UB) ligase, E6 stirs the protein ubiquitination pathways toward the oncogenic transformation of the infected cells. For a systematic measurement of E6 reprogramming of the substrate pool of E6AP, we performed a proteomic screen based on “orthogonal UB transfer (OUT)” that allowed us to identify the ubiquitination targets of E6AP dependent on the E6 protein of HPV‐16, a high‐risk viral subtype for the development of cervical cancer. The OUT screen identified more than 200 potential substrates of the E6‐E6AP pair based on the transfer of UB from E6AP to the substrate proteins. Among them, we verified that E6 would induce E6AP‐catalyzed ubiquitination of importin proteins KPNA1‐3, protein phosphatase PGAM5, and arginine methyltransferases CARM1 to trigger their degradation by the proteasome. We further found that E6 could significantly reduce the cellular level of KPNA1 that resulted in the suppression of nuclear transport of phosphorylated STAT1 and the inhibition of interferon‐γ‐induced apoptosis in cervical cancer cells. Overall, our work demonstrates OUT as a powerful proteomic platform to probe the interaction of E6 and host cells through protein ubiquitination and reveals a new role of E6 in down‐regulating nuclear transport proteins to attenuate tumor‐suppressive signaling. 

Glycosyltransferase OGT catalyzes the conjugation of O-linked β-D-N-acetylglucosamine (O-GlcNAc) to Ser and Thr residues of the cellular proteins and regulates many key processes in the cell. Here, we report the identification of OGT as a ubiquitination target of HECT-type E3 ubiquitin (UB) ligase E6AP, whose overexpression in HEK293 cells would induce the degradation of OGT. We also found that the expression of E6AP in HeLa cells with the endogenous expression of the E6 protein of the human papillomavirus (HPV) would accelerate OGT degradation by the proteasome and suppress O-GlcNAc modification of OGT substrates in the cell. Overall, our study establishes a new mechanism of OGT regulation by the ubiquitin–proteasome system (UPS) that mediates the crosstalk between protein ubiquitination and O-GlcNAcylation pathways underlying diverse cellular processes. 

Ubiquitin and ubiquitin like proteins (UBLs) play key roles in eukaryotes. These proteins are attached to their target proteins through an E1-E2-E3 cascade and modify the functions of these proteins. Since the discovery of ubiquitin, several UBLs have been identified, including Nedd8, SUMO, ISG15, and Atg8. Ubiquitin and UBLs share a similar three-dimensional structure: β -grasp fold and an X-X-[R/A/E/K]-X-X-[G/X]-G motif at the C-terminus. We have previously reported that ubiquitin, Nedd8, and SUMO mimicking peptides which all contain the conserved motif X-X-[R/A/E/K]-X-X-[G/X]-G still retained their reactivity toward their corresponding E1, E2, and E3 enzymes. In our current study, we investigated whether such C-terminal peptides could still be transferred onto related pathway enzymes to probe the function of these enzymes when they are fused with a protein. By bioinformatic search of protein databases, we selected eight proteins carrying the X-X-[R/A/E/K]-X-X-[G/X]-G motif at the C-terminus of the β -grasp fold. We synthesized the C-terminal sequences of these candidates as short peptides and found that three of them showed significant reactivity with the ubiquitin E1 enzyme Ube1. We next fused the three reactive short peptides to three different protein frames, including their respective native protein frames, a ubiquitin frame and a peptidyl carrier protein (PCP) frame, and measured the reactivities of these peptide-fused proteins with Ube1. Peptide-fused proteins on ubiquitin and PCP frames showed obvious reactivity with Ube1. However, when we measured E2 UbcH7 transfer, we found that the PCP-peptide fusions lost their reactivity with UbcH7. Taken together, these results suggested that the recognition of E2 enzymes with peptide-fused proteins depended not only on the C-terminal sequences of the ubiquitin-mimicking peptides, but also on the overall structures of the protein frames.