The Molecular Mechanisms And Physiological/Pathological Roles Of REGγin P53Degradation

REGy (also known as PA28y,11Sγ, or PSME3) was first identified as the Ki antigen, a nuclear protein targeted by autoantibodies found in sera of patients with systemic lupus erythematosus. As a proteasome activator, REGy has been known that it can bind with the20S core proteasome and degrade some intact proteins in a ubquitin-and ATP-independent manner, such as SRC3, p21, p19and HCV core protein. Moreover, REGy plays important physiological roles in mammals. Studies have shown that REGy knock-out mice display modest growth retardation and have a deficiency in immune system. However, many other physiological functions of REGy have not been well known. In this study, we do some exploration on REGy’s other functions based on REGγ knock-out mouse model, combined with cellular experiments.Firstly, this study found that REGγ can regulate p53protein level and its down-steam factors in A549cells, as well as its cellular distribution. Our study showed that REGy can promote p53’s translocation from nucleus to cytoplasm through enhancing its mono-ubiquitination by MDM2. Further studies found that the regulation of p53’s cellular distribution by REGy is MDM2dependent. When MDM2’s expression was inhibited by RNA knockdown experiment, p53’s cellular distribution was not affected by REGy anymore.Secondly, REGy was found highly expressing in mice testis during detection of REGy’s expression in different organs of mice by western blot; Meanwhile, when wild type female mice were crossed with REGγ knock-out male mice or wild type male mice, the littermate number of female mice crossed with REGγ knock-out female mice was smaller than that crossed with wild type male mice; The analysis results of HTM-OVIS revealed that the sperm concentration and the sperm activity of REGy knock-out male mice decreased; Also, the ability of REGy knock-out male mice to impregnate wild type female mice was decreased comparing to wild type mice. Besides, this study focused on the mechanisms that REGy play roles in male reproduction through studies about spermatogonial stem cells(SSCs) and primary spermatocytes. On one hand, we found deletion of REGy in male mice up-regulates p53expression in testis, and p53’s high expression can ultimately lead to decrease of SSCs through its inhibiting effect on PLZF transcription, which is the marker protein of SSCs and plays an important role in maintaining SSCs self-renewal. On the other hand, REGγ knock-out mice display more apoptotic primary spermatocytes in testis, and this is mediated by high expression of p53protein when REGγ was depleted.Finally, this study found REGy plays an important role in liver cancer initiation besides its role in male reproduction. When the wild type and REGγ knock-out male mice were injected with DEN, a drug that can induce the initiation of live cancer, the REGγ knock-out male mice showed a delay in tumor appearance, and the tumor size and tumor number also decreased comparing to the wild type mice. Very likely, the resistance to liver cancer initiation when injected with DEN is caused by the high expression of p53in REGγ knock-out male mice.In summary, this study found REGγ can regulate p53’s level and its cellular distribution and play important roles in regulation of male reproduction and liver cancer initiation. Also, the mechanisms of REGγ functions were detailed explored. These discoveries will widen the new research areas of REGγ.