SENP3-mediated De-SUMOylation Of P53 Inhibits Its Transcriptional Activity In Lung Cancer

p53 is an important stress sensor and tumor-suppressor.In resting state of the cells,p53 rapidly degrades and keeps low level,while exposure of internal or external stimulus,such as ionizing irradiation and oncogene activation,p53 accumulates,activates and promotes the transcription of downstream genes which leads to DNA damage repair or apoptosis induction to eliminate potential malignant cells.In tumorigenesis,p53 gene mutation is one of prominent phenomena,81% occurrence in lung cancer patients.However,it is unclear why normal expression of wild-type p53 bears low activity in some tumor cells.This study mainly explored potential mechanism by which transcriptional activity of p53 is inhibited.A number of studies demonstrated different modes of p53 activity regulation,especially posttranslational modifications such as ubiquitin,phosphorylation,acetylation and SUMOylation.Reversible and dynamic SUMOylation is a new kind of ubiquitin-like modification,regulating stability,localization and activity of many substrate proteins.It has been reported that SUMOylation could directly or indirectly modulate the activity of p53,but it is not consistent in the effects of this modification on p53 transcriptional activity in diverse research systems.Our previous studies have found SUMO protease SENP3 is also stress sensor through de-SUMO2/3 of a variety of specific substrates.We also found that SENP3 levels in many tumor tissues are higher than para-carcinoma tissues and normal tissues.At the same time,the tumor database or protein atlas shows SENP3 expression is also higher in the some cells bearing low p53 activity such as lung cancer cell line A549.To sum up,this research intends to explore whether SENP3 mediates de-SUMO2/3 modification of p53 and thus inhibits its activity.Answering this question may provide possible explanation for the inactivation of p53 in some tumor cells.In this study,lung cancer cell lines A549(p53 wild type)and H1299(p53 null)were used.The study was divided into three parts.In the first part,SUMO2/3 modification and SUMO conjugation site of p53 was verified and whether SENP3 interacted with p53 and mediated de-SUMOylation of p53 under oxidative stress was demonstrated.Through co-immunoprecipitation and Ni-nitrilotriacetic acid resin pull-down technology SUMO2/3 modification of p53 lysine 386 were proved and SENP3/p53 interaction enhancement in response to oxidative stress was explored.In the second part,whether the nuclear localization of p53 was altered by SUMOylation and de-SUMOylation was observed.Through immunofluorescence technology,we found SENP3-mediated de-SUMOylation of p53 did not result in translocation of nuclear p53 under resting state or oxidative stress.In the third part,whether p53 transcription activity was inhibited by SENP3-mediated de-SUMOylation was explored.By comparing the effects of p53 wild-type and SUMOylation site mutants,we found that de-SUMO2/3 modification of p53 specifically inhibited target gene p21 expression and promoted cell proliferation.