The Role Of Regy In The Degradation Of Oxidized Proteins And The Study On Other REGγ’s Target Protein

Oxidized proteins,which is closely related to aging and neurodegenerative diseases,are formed and accumulated under the oxidative stress,disordering the cellular function. The oxidative damage of some important regulators,such as p21,will disorder the cell balance. And the hemoglobin which has vital biological function is easily damaged by oxidative stress.So,the degradation of the oxidative damaged proteins is important to normal cellular activities.The proteasome plays a vital role in the degradation of the oxidized proteins which is toxic to the cells.Here,we demonstrated that REGy,a 11S proteasome activator,played a role in the degradation of oxidized p21,and we also did research ahout REGy’s regulation on hemoglobin.In HeLa and H1299,the half life of p21 was shortened under the H2O2 treatment,and the decrease of p21 protein level in the early stage wss mRNA-independent.Then we demonstrated that REGy promoted the degradation of oxidized p21 under treatment of 250μM/2mM H2O2 or 5mM/20mM PHZ.And we got the consistent results in A549 shN&shR,HepG2,shN&shR and MEF REGy+/-®y-/-, indicating that REGy’s function in degradation of oxidized proteins is conserved to a certain extent.We also demonstrated that the degradation of p21 under oxidative stress is regulated by the changes in redox state. About the mechanism,we demonstrated that the degradation of oxidized p21 was proteasome-dependent.And the H2O2 stress increased the REGy-induced proteasome trypsin-like activity,also increased the interaction between REGy and 20 S to form more REGy-proteasome.These increased activities induced by oxidative stress contributed to the degradation of oxidized p21 or other potential targets to a certain extent.Hemoglobin 8(HBD) was one potential target identified in the Mass Spectrometry assay detecting the targets of REGy.And it had been reported that the oxidative stress induced damage of hemoglobins can be degraded in an ATP-independent pathway. We constructed the HBD plasmid for cell transfection.In HeLa,compared with overexpression of REGy wt,overexpression of REGy N151Y(losing normal function) contributed to the accumulation of HBD. The immunofluorescence result showed that HBD level was higher in HeLa co-transfected with REGy N151Y than co-transfected with REGywt.Also we got the phenomenon of colocalization of HBD and REGy.And in HeLa REGy knockdown stable cell lines,we detected that REGy promoted the decline of HBD under oxidative stress.In mouse bone marrow,liver and spleen which are related to hemoglobin regeneration,the REGy-/-tissues had a higher level of hemoglobin than REGy-/-tissues,which was consistent with the HBD results in HeLa cell.These results indicated REGy can regulate some hemoglobin subunits directly or indirectly.But it needs more research on the mechanism of REGy’s regulation on hemoglobin.Taken together, our studies demonstrate that REGy modulates oxidative stress-mediated p21 degradation in the early stage via promoting 20S proteasome activity.It highlights a new mechanism model of oxidized protein degradation and a new field to study cell oxidative stress response.The role of REGy in the degradation of oxidized proteins contributes to mechanism of aging and ROS related diseases.And the regulation of REGy on hemoglobin also contributes to the theory of development of hemoglobin diseases.