Construction And Application Of REGγ-dependent Protein Degradation System

The proteasome degradation system is one of the most important protein degradation systems. The proteasome exist in both cytoplasm and nucleus, and its activity is central to many cellular functions including protein quality control, DNA repair, transcription, cell-cycle regulation, signal transduction, and antigen presentation. The proteasome is composed of two parts:20s proteasome and activators which includes 19s,11s and PA200. REGγis one of the 11s family, it can active the trypsin-like activity of the 20s proteasome.REGγjust can degrade some peptides come from the degradation by others activators in traditional views. Xiaotao Li etc. found that REGγcan degrade SRC-3 in cells in 2006. It is the first time to be proved can degrade intact protein in cell. From that on, more proteins that could be degraded by REGγwere found including p21, p19, MDM2 and HCV. Those data indicate REGγplays an important role in many biological processes.REGγcan degrade proteins is one mechanism for those physiological processes it plays. So to find proteins be degraded by REGγis an important research field. For this aim, we purified 20s proteasome and REGγby different HPLC methods, for examples:ion exchange, size exclusion and affinity purification. We combined purified 20s proteasome and REGγtogether to construct an in vitro degradation system after checked the aggregation of the two proteins. Then we used p21 protein to test this system. What's more, for the first time, we also proved HCV can be degraded by REGy in vitro. All those data proved the activity of this system. Base on this system we can detect the potential target proteins that degrade by REGy easily, and it is also useful in the research of protein modification.