The Mechanism Of Degradation Of Oxidative HCV Core Protein And P21 By REGγ

During the oxidative stress from oxidants in vitro and in vivo, there are series of changes in cells and make reactive oxide species(ROS). ROS is a natural by-product of living metabolism, excess of ROS will cause damages to proteins, nuclear acids and cell membrane. Excessive oxidative stress may induce some diseases, like:Atherosclerosis, Pulmonary Fibrosis, cancer, Neuro-degeneration disease and aging.According to previous studies, oxidative damage directly targets proteins and the odd of ROS interacting with proteins is 20 times of that with nuclear acids. Therefore, the study on the mechanism of anti-oxidative stress in vivo focus on oxidative protein level. Intracellular anti-oxidative damage mechanism is supposed to cleave excessive oxidative proteins. It is reported that 20S proteasome plays an important role in degradation of oxidative proteins, and the 1 IS regulator (REGy) of proteasome involves in regulating the proteolysis by 20S proteasome independent of ATP and ubiquitin.In our study, the protein level of p21 in HeLa cells rapidly decreased after treatment of H2O2, while the mRNA level didn’t change. When the REGy-stable knockout HeLa cells treated with H2O2, the p21 protein level had little change. The same results could be got under different concentrations and kinds of oxidants, it proves that REGy is critical for proteolysis of oxidative p21 protein. In order to get further study, we chose another REGy target protein-HCV core protein. We constructed the expression vector of HCV core, then transfected it into HeLa shN(control) and shREGy cells, those cells were treated with oxidants during time course. The Western Blotting results shared the same trends with p21 protein’s. We also used the REGy-dysfunction and the normal REGy cell lines, the results showed that oxidative HCV core protein in the normal REGy cell lines was degraded dependent of REGy, whereas in the REGy-dysfunction cell lines no significant changes were observed. That proved the critical role of REGy during the cleavage of oxidative proteins. Through the degradation in vitro,Co-IP (Co-Immunoprecipitation) and trypsin-like activity experiments, it revealed that:excessive oxidative stress could increase the trypsin-like activity of 20S proteasome and enhance the interaction of REGy and 20S proteasome. On the other hand, we detected the apoptosis marker and the cellular morphology of cells during oxidative stress. Results proved that the degradation of oxida-tive protein p21 and HCV core protein didn’t have direct relationship with apoptosis caused by oxidative stress, which is a good supplement for the mechanism of cleavage of oxidative proteins.Though the mechanism of cleavage of oxidative proteins is not clear, which still need further studies. The approaches and results of this paper will not only put up with a novel pathway for study of proteolysis of oxidative proteins, but also provide referred theory for researches on oxidative-related diseases.