Summary The p53 pro-apoptotic tumor suppressor is mutated or functionally altered in most cancers. In epithelial tumors induced by “high-risk” mucosal Human Papillomaviruses (hrm-HPVs), including human cervical carcinoma and a growing number of head-and-neck cancers 1, p53 is degraded by the viral oncoprotein E6 2. In this process, E6 binds to a short LxxLL consensus sequence within the cellular ubiquitin ligase E6AP 3. Subsequently, the E6/E6AP heterodimer recruits and degrades p53 4. Neither E6 nor E6AP are separately able to recruit p53 3,5, and the precise mode of assembly of E6, E6AP and p53 is unknown. Here, we solved the crystal structure of a ternary complex comprising full-length HPV16 E6, the LxxLL motif of E6AP and the core domain of p53. The LxxLL motif of E6AP renders the conformation of E6 competent for interaction with p53 by structuring a p53-binding cleft on E6. Mutagenesis of critical positions at the E6-p53 interface disrupts p53 degradation. The E6-binding site of p53 is distal from previously described DNA- and protein-binding surfaces of the core domain. This suggests that, in principle, E6 may avoid competition with cellular factors by targeting both free and bound p53 molecules. The E6/E6AP/p53 complex represents a prototype of viral hijacking of both the ubiquitin-mediated protein degradation pathway and the p53 tumor suppressor pathway. The present structure provides a framework for the design of inhibitory therapeutic strategies against HPV-mediated oncogenesis.

Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this aim, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to a thousand domain-motif equilibrium binding affinities per day. Extracts of overexpressed domains are incubated with peptide-coated resins and subjected to filtration. Binding affinities are deduced from microfluidic capillary electrophoresis of flow-throughs. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from Human Papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human PDZome. We obtained exquisite sequence-dependent binding profiles, describing quantitatively the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has a wide potential for quantifying the specificities of interactomes.

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.

Understanding the mechanisms of coronavirus disease 2019 (COVID-19) disease severity to efficiently design therapies for emerging virus variants remains an urgent challenge of the ongoing pandemic. Infection and immune reactions are mediated by direct contacts between viral molecules and the host proteome, and the vast majority of these virus–host contacts (the ‘contactome’) have not been identified. Here, we present a systematic contactome map of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with the human host encompassing more than 200 binary virus–host and intraviral protein–protein interactions. We find that host proteins genetically associated with comorbidities of severe illness and long COVID are enriched in SARS-CoV-2 targeted network communities. Evaluating contactome-derived hypotheses, we demonstrate that viral NSP14 activates nuclear factor κB (NF-κB)-dependent transcription, even in the presence of cytokine signaling. Moreover, for several tested host proteins, genetic knock-down substantially reduces viral replication. Additionally, we show for USP25 that this effect is phenocopied by the small-molecule inhibitor AZ1. Our results connect viral proteins to human genetic architecture for COVID-19 severity and offer potential therapeutic targets.

Influenza A viruses (IAVs) are responsible for mild-to-severe seasonal respiratory illness of public health concern worldwide, and the risk of avian strain outbreaks in humans is a constant threat. Elucidating the requisites of IAV adaptation to humans is thus of prime importance. In this study, we explored how PB2 replication proteins of IAV strains with different levels of virulence in humans hijack a major protein modification pathway of the human host cell, the ubiquitin proteasome system (UPS). We found that the PB2 protein engages in an extended interplay with the UPS that evolved along with the virus’s adaptation to humans. This suggests that UPS hijacking underlies the efficient infection of humans and can be used as an indicator for evaluation of the potential of avian IAVs to infect humans. Several UPS factors were found to be necessary for infection with circulating IAV strains, pointing to potential targets for therapeutic approaches.

The E6 oncoproteins of high-risk mucosal (hrm) HPVs contain a pocket that captures LxxLL motifs and a C-terminal motif that recruits PDZ domains, both functions being crucial for HPV-induced oncogenesis. Here, we fused together a PDZ domain and a LxxLL motif both previously known to bind E6. Using NMR, calorimetry and mammalian protein complementation assay, we show that the resulting PDZ-LxxLL chimera is a bivalent nanomolar ligand of E6, while its separated PDZ and LxxLL components are only micromolar binders. The chimera binds to all hrm-HPV E6 proteins tested but not to low-risk mucosal or cutaneous HPV E6. Adenovirus-mediated expression of the chimera specifically induces the death of HPV-positive cells, concomitant with increased levels of tumour suppressor P53, of its transcriptional target p21, and of apoptosis marker cleaved caspase 3. The bifunctional PDZ-LxxLL chimera opens new perspectives both for diagnostics and therapy of HPV-induced cervical and head and neck cancers.

The E6 oncoproteins of high-risk mucosal (hrm) human papillomaviruses (HPVs) contain ap ocket that captures LxxLL motifs and aC-terminal motif that recruits PDZ domains,w ith both functions being crucial for HPV-induced oncogenesis.Achimeric protein was built by fusing aP DZ domain and an LxxLL motif,b oth knownt ob ind E6. NMR spectroscopy, calorimetry and am ammalian protein complementation assayc onverged to showt hat the resulting PDZLxxLL chimera is abivalent nanomolar ligand of E6, while its separated PDZ and LxxLL components are only micromolar binders.The chimera binds to all of the hrm-HPV E6 proteins tested but not to low-risk mucosal or cutaneous HPV E6. Adenovirus-mediated expression of the chimera specifically induces the death of HPV-positive cells,c oncomitant with increased levels of the tumour suppressor P53, its transcriptional target p21, and the apoptosis marker cleaved caspase 3. The bifunctional PDZ-LxxLL chimera opens new perspectives for the diagnosis and treatment of HPV-induced cancers.Papillomaviruses (PVs) infect the cutaneous and mucosal epithelia of vertebrates.[1] Whereas most PVs are benign, asubset of "high-risk" mucosal human PV types (hrm-HPVs) induce cervical cancer [2] and as ignificant proportion of head and neck cancers.[3] HPV 16 is the most prevalent and best studied hrm-HPV type.H PV carcinogenesis is primarily linked to two PV oncoproteins,E 6a nd E7.[4] hrm-HPV E6 recruits the ubiquitin ligase E6AP and the tumour suppressor P53, which leads to ubiquitin-mediated degradation of P53. [5] This dramatically reduces P53 protein levels in HPV-infected cells,t hereby disrupting the pro-apoptotic and genome watchdog functions of P53. In this process,E 6b inds within E6AP an acidic leucine-rich motif containing an LxxLL consensus sequence.[6] TheX-ray crystallography structure of the E6/E6AP complex has shown that E6 captures the motif within ac onserved basic-hydrophobicp ocket.[7] Hrm-HPV E6 also binds to and sometimes promotes the degradation of several PDZ-containing cellular proteins,w hich regulate cell-cell adhesion, cell polarity,a nd apoptosis. Hrm-HPV E6 captures PDZ domains by means of as hort PDZ binding motif (PBM), which is situated at the extreme Cterminus of E6.[8] Several structures of E6/PDZ complexes have been solved [9] . E6 thus possesses two interaction surfaces responsible for its two best-described oncogenic activities.W ee xploited our recent structural insights into both activities [7, 9a] to build ah eterobivalent E6-binding construct to simultaneously target both interaction surfaces.W ed esigned ac himeric PDZ-LxxLL fusion protein ( Figure S1 in the Supporting Information) comprising the MAGI1 PDZ2 domain (the second of the six PDZ domains of MAGI1, sometimes named "PDZ1" or "PDZ2/6" in earlier works), [9a] at hree-alanine linker,and the E6-binding LxxLL motif of E6AP (sequence: ELTLQELLGEER).[7] By combining our previous structures of the E6/LxxLL complex (PDB ID 4GIZ [7] ;F igure 1a)a nd of the E6/PDZ2 complex (PDB ID 2KPL[9a] ;F igure...

Targeted protein degradation (TPD) strategies exploit bivalent small molecules to bridge substrate proteins to an E3 ubiquitin ligase to induce substrate degradation. Few E3s have been explored as degradation effectors due to a dearth of E3-binding small molecules. We show that genetically induced recruitment to the GID4 subunit of the CTLH E3 complex induces protein degradation. An NMR-based fragment screen followed by structure-guided analog elaboration identified two binders of GID4, 16 and 67, with K d values of 110 and 17 μM in vitro. A parallel DNA-encoded library (DEL) screen identified five binders of GID4, the best of which, 88, had a K d of 5.6 μM in vitro and an EC50 of 558 nM in cells with strong selectivity for GID4. X-ray co-structure determination revealed the basis for GID4–small molecule interactions. These results position GID4-CTLH as an E3 for TPD and provide candidate scaffolds for high-affinity moieties that bind GID4.