Design of a PROTAC that antagonizes and destroys the cancer-forming X-protein of the hepatitis B virus

The X-protein of the hepatitis B virus (HBV) is essential for virus infection and contributes to the development of HBV-induced hepatocellular carcinoma (HCC), a disease which causes more than one million deaths each year. Here we describe the design of a novel PROTAC (proteolysis targeting chimeric molecule) capable of simultaneously inducing the degradation of the X-protein, and antagonizing its function. The PROTAC was constructed by fusing the N-terminal oligomerization and C-terminal instability domains of the X-protein to each other, and rendering them cell-permeable by the inclusion of a polyarginine cell-penetrating peptide (CPP). It was predicted that the oligomerization domain would bind the X-protein, and that the instability domain would cause the X-protein to be targeted for proteasomal degradation. Addition of the PROTAC to HepG2 liver cancer cells, engineered to express full-length and C-terminally truncated forms of the X-protein, resulted in the degradation of both forms of the X-protein. A cell-permeable stand-alone form of the oligomerization domain was taken up by HepG2 cells, and acted as a dominant-negative inhibitor, causing inhibition of X-protein-induced apoptosis. In summary, the PROTAC described here induces the degradation of the X-protein, and antagonizes its function, and warrants investigation in a preclinical study for its ability to prevent or treat HBV infection and/or the development of HCC.     

STD and TRNOESY NMR studies for the epitope mapping of the phosphorylation motif of the oncogenic protein beta-catenin recognized by a selective monoclonal antibody

The interaction of the P-beta-Cat(19-44) peptide, a 26 amino acid peptide (K(19)AAVSHWQQQSYLDpSGIHpSGATTTAP(44)) that mimics the phosphorylated beta-Catenin antigen, has been studied with its monoclonal antibody BC-22, by transferred nuclear Overhauser effect NMR spectroscopy (TRNOESY) and saturation transfer difference NMR (STD NMR) spectroscopy. This antibody is specific to diphosphorylated beta-Catenin and does not react with the non-phosphorylated protein. Phosphorylation of beta-Catenin at sites Ser33 and Ser37 on the DSGXXS motif is required for the interaction of beta-Catenin with the ubiquitin ligase SCF(beta-TrCP). beta-TrCP is involved in the ubiquitination and proteasome targeting of the oncogenic protein beta-Catenin, the accumulation of which has been implicated in various human cancers. The three-dimensional structure of the P-beta-Cat(19-44) in the bound conformation was determined by TRNOESY NMR experiments; the peptide adopts a compact structure in the presence of mAb with formation of turns around Trp25 and Gln26, with a tight bend created by the DpS(33)GIHpS(37) motif; the peptide residues (D32-pS37) forming this bend are recognized by the antibody as demonstrated by STD NMR experiments. STD NMR studies provide evidence for the existence of a conformational epitope containing tandem repeats of phosphoserine motifs. The peptide's epitope is predominantly located in the large bend and in the N-terminal segment, implicating bidentate association. These findings are in excellent agreement with a recently published NMR structure required for the interaction of beta-Catenin with the SCF(beta-TrCP) protein.     

ASB proteins interact with Cullin5 and Rbx2 to form E3 ubiquitin ligase complexes

The ankyrin repeat and SOCS box (ASB) family is composed of 18 proteins from ASB1 to ASB18 and belongs to the suppressor of cytokine signaling (SOCS) box protein superfamily. ASB2 was recently shown to interact with a certain Cul-Rbx module to form an E3 ubiquitin (Ub) ligase complex, but the functional composition of the ASB-containing E3 Ub ligase complexes remains to be characterized. Here, we show that ASB proteins interact with Cul5-Rbx2 but neither Cul2 nor Rbx1 in cells. Mutational analysis revealed that the highly conserved amino acid sequences of the BC box and Cul5 box in the SOCS box of ASB proteins were essential for the interaction with Cul5-Rbx2. Although ASB proteins show slight divergences from the consensus sequences of the BC box and Cul5 box, all five tested ASB proteins bound to Cul5-Rbx2. Furthermore, all three tested ASB complexes containing Cul5-Rbx2 were found to have E3 Ub ligase activity. These findings suggest that the ASB family proteins interact with Cul5-Rbx2 to form E3 Ub ligases and play significant roles via a ubiquitination-mediated pathway.