Mapping the interactome of HPV E6 and E7 oncoproteins with the ubiquitin‐proteasome system

Abstract

Protein ubiquitination and its reverse reaction, deubiquitination, regulate protein stability, protein binding activity, and their subcellular localization. These reactions are catalyzed by the enzymes E1, E2, and E3 ubiquitin (Ub) ligases and deubiquitinases (DUBs). The Ub-proteasome system (UPS) is targeted by viruses for the sake of their replication and to escape host immune response. To identify novel partners of human papillomavirus 16 (HPV16) E6 and E7 proteins, we assembled and screened a library of 590 cDNAs related to the UPS by using the Gaussia princeps luciferase protein complementation assay. HPV16 E6 was found to bind to the homology to E6AP C terminus-type Ub ligase (E6AP), three really interesting new gene (RING)-type Ub ligases (MGRN1, LNX3, LNX4), and the DUB Ub-specific protease 15 (USP15). Except for E6AP, the binding of UPS factors did not require the LxxLL-binding pocket of HPV16 E6. LNX3 bound preferentially to all high-risk mucosal HPV E6 tested, whereas LNX4 bound specifically to HPV16 E6. HPV16 E7 was found to bind to several broad-complex tramtrack and bric-a-brac domain-containing proteins (such as TNFAIP1/KCTD13) that are potential substrate adaptors of Cullin 3-RING Ub ligases, to RING-type Ub ligases implicated in innate immunity (RNF135, TRIM32, TRAF2, TRAF5), to the substrate adaptor DCAF15 of Cullin 4-RING Ub ligase and to some DUBs (USP29, USP33). The binding to UPS factors did not require the LxCxE motif but rather the C-terminal region of HPV16 E7 protein. The identified UPS factors interacted with most of E7 proteins across different HPV types. This study establishes a strategy for the rapid identification of interactions between host or pathogen proteins and the human ubiquitination system. Abbreviations APC/C, anaphase-promoting complex/cyclosome; BTB, broad-complex tramtrack and bric-a-brac; CRL, Cullin-ring ubiquitin ligase; DUB, deubiquitinase; DWD, DBD1-binding W40 protein; Gluc1, Gaussia luciferase fragment 1; Gluc2, Gaussia luciferase fragment 2; Gluc, Gaussia luciferase; GPCA, Gaussia protein complementation assay; HECT, homology to E6AP C terminus; HPV, human papillomavirus; IQR, interquartile range; JAMM, Jab1/mov34/Mpr1 Pad1 N-terminal+; MJD, Machado/Josephin domain; NLR, normalized luminescence ratio; OTU, ovarian tumor protease; PBM, PDZ-binding motif; PPI, protein-protein interaction; PRS, positive reference set; RING, really interesting new gene; RLU, relative luciferase unit; RRS, random reference set; SOCS, suppressor of cytokine signaling; SRF, substrate recognition factor; Ubl, ubiquitin-like; Ub, ubiquitin; UCH, ubiquitin C-terminal hydrolase; UPS, ubiquitin-proteasome system; USP, ubiquitin-specific protease.

HPV E6 and E7 oncoproteins cooperatively alter the expression of Disc Large 1 polarity protein in epithelial cells

“…This result suggests that E7 may increase DLG1 protein levels probably by contributing to its stabilization and/or preventing its degradation. In line with this, it was recently reported that HPV E7 interacts with different components of the ubiquitinproteasome system including proteins that reverse the ubiquitination process, and, in this way, E7 might interfere with the degradation of certain cellular substrates [42]. It is also possible that E7 induced signal pathways that drive changes in DLG1 posttranslational modifications, rendering it more resistant to proteasomal degradation.…”

HPV Oncoproteins and the Ubiquitin Proteasome System: A Signature of Malignancy?

“…These studies assembled and screened a library of 590 cDNAs related to the UPS that covered about 50% of the human ubiquitination system, together with co-immunoprecipitation to confirm novel cell target protein-E6 interaction. Results indicated various new target proteins, including three RING-type Ub ligases MGRN1, LNX3 and LNX4 [38,103]. MGRN1 was shown to interact with E6 proteins from LR HPV-6 and HR HPV types 16, 18 and 33, as well as β-types HPV types 8 and 38.…”

“…Ubiquitin is transferred to target proteins by the ubiquitination cascade: first, ubiquitin is activated and bound by an E1 ubiquitin-activating enzyme, then transferred to an E2 ubiquitin-conjugating enzyme and, finally, transferred to lysine residues on the target protein by E3 ubiquitin-protein ligase [36,37]. So far, studies have identified two E1 activating enzymes, 40 E2 conjugating enzymes, around 800 E3 ubiquitin-protein ligases and about 100 DUBs [38]. While all components of the UPS are of great importance for ubiquitination, individual ubiquitin-protein ligases may be valuable viral targets as they are involved in substrate recognition, and hence define the specificity of the system.…”

“…However, it still remains to be clarified whether BRCA1 plays a role in the initiation of cervical cancer. In a recent study, Poirson et al [38] using the Gaussia princeps luciferase protein complementation assay (GPCA), identified a number of cellular ubiquitin ligases or substrates with ubiquitin ligase activity as interacting partners of HPV E6 [38]. These studies assembled and screened a library of 590 cDNAs related to the UPS that covered about 50% of the human ubiquitination system, together with co-immunoprecipitation to confirm novel cell target protein-E6 interaction.…”

Viral oncoproteins and ubiquitination: accessing a cellular toolbox for modifying protein function

“…In this issue of The FEBS Journal, Poirson et al . have made a global examination of the interactions of the two major human papillomavirus (HPV) oncoproteins, E6 and E7, with components of the UPS. Over 200 different HPV types exist, but the major interest in this group of viruses stems from the fact that a small number of HPV types are major causes of human cancer, with cervical cancer being the most frequent .…”