Study On Mechanism Of HCK NS4B Interact With Scribble And Hippo Pathway

ObjectiveHepatitis C virus (HCV) is an enveloped positive single-stranded virus and it is one of the major causes of liver related diseases. We have previously predicted possible interaction motifs within NS4B using basic ELM software and found that at the C-terminus of NS4B contains a PDZ-binding motif (PBM) which could bind cellular PDZ-containing proteins such as the membrane protein, Scribble. In this study we aimed to investigate the interaction characters of NS4B and Scribble and the affection of NS4B to the Scribble protein including the protein expression level or relocation. On the other hand, we detected the role of the NS4B in the Hippo pathway.MethodsIn this work, series of plasmids were constructed. Immunofluorescence microscopy assay was used for investigating the co-location of NS4B and Scrbble protein. Co-immunoprecipitation assay was used to detect the interaction of NS4B and Scrbble. To detecte the proteins level, western-blot was carried out. Cloning formation assay was also involved to research the transformation activity of HEPG2cells with NS4B stimulated. At last, Oil red0test was taken for investigating the lipid drops synthesis.ResultsIn this study, both immunofluorescence and co-immunoprecipitation assays demonstrated that NS4B PBM interacts with PDZ-containing tumor suppressor protein, Scribble and this interaction requires at least three PDZ domains of Scribble. In addition, we found that NS4B PBM activates the proteasome pathway to specifically degrade the Scribble protein level as NS4B significantly increased the ubiquitination level of Scribble and reduced the protein level of ubiquitin specific peptidase14(Usp14). Furthermore, NS4B PBM mutants showed reduced transformation capacity compared with full length NS4B, indicating that NS4B PBM facilitates NS4B-mediated hepatoma cells transformation. Finally, We still found that NS4B mediated the gene expression level of Merlin. Merlin significantly decreased the lipid drops synthesis; on the contrary, NS4B increased the lipid drops synthesis obviously.ConclusionHere, we found that NS4B PBM is a key motif that enables the NS4B to interact with Scribble protein and at least three PDZ domains of Scribble are needed for the association of these two proteins. Furthermore, the interaction between NS4B and Scribble decreased the protein levels of Scribble through the proteasome pathway and NS4B PBM domain is required for NS4B-mediated Scribble degradation. In addition, NS4B PBM contributes to the ability of NS4B to transform hepatoma cells. Furthermore, NS4B also mediate the lipid drops synthesis via the Hippo pathway. Taken together, our study provides a possible mechanism by which NS4B contributes to HCV pathogenesis by targeting tumor suppressor protein, Scribble for degradation.