Targeting the Two Oncogenic Functional Sites of the HPV E6 Oncoprotein with a High‐Affinity Bivalent Ligand

Abstract

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...

Affinity and specificity of motif-based protein–protein interactions

“…Viral proteins often contain multiple motifs used to hijack different human protein-protein interactions [63][64][65]. In such cases the avidity effect could potentially be explored by designing multivalent inhibitors towards the viral protein by combining parts of the different human targets in a "protein drug" [66,67].…”

Recognition of high-risk HPV E6 oncoproteins by 14-3-3 proteins studied by interactomics and crystallography

“…In HPV-transformed cells, E6 participates in counteracting apoptosis, altering differentiation pathways, polarity and adhesion properties, and thereby sustains cell proliferation 1,7 . Inhibition of E6 in HPV-positive cell lines results in the cell growth arrest and induces apoptosis or rapid senescence 8,9,10,11 . E6 has been found to interact with numerous distinct cellular target proteins involved in a variety of cellular functions 12 .…”

Artificial microRNAs against the viral E6 protein provoke apoptosis in HPV positive cancer cells

“…Another possibility would be the use of a combinatorial treatment with Ad18_2 and Cisplatin[8]. Very recently we designed a high affinity bivalent ligand able to bind specifically to E6 of all high risk HPV[21]. The adenovirus-driven expression of this ligand provokes apoptosis of HeLa and SiHa cells in vitro, and remains to be tested in vivo in conjunction with the vectors reported in this work.…”