Mechanism Study On Ubc9-mediated SUMOylation Against Oxidative Stress Derived ROS Production In Pancreatic ? Cell

Backgroud: Previous studies have indicated that oxidative stress plays a key role in pancreatic ? cell dysfunction and apoptosis.NADPH oxidase is one of the main sources of ROS production.SUMOylation has been reported to inhibit NADPH oxidase derived ROS generation.Our study group also revealed that SUMOylation depletion results in ROS accumulation and ? cell dysfunction in islets of ? cell specific Ubc9 gene knockout mice,which indicated the vital role of SUMOylation in modulating oxidative response and in keeping ? cell functioning properly.p47 serves as the organizer of NADPH oxidase complex,its activation and translocation to cell membrane is the key step leading to ROS generation.Bioinformatic analysis of p47 highly suggested the role of which as a potential SUMOylation target.Thus,we hypothesis that SUMO1 modulates NADPH oxidase derived ROS generation via interacting with p47.Objective: To find out the SUMO-binding substrate in pancreatic ? cell under oxidative stimulation,by which SUMO modulates ROS production.And further examine the location of the binding motifs at the target protein.Methods: Overexpression of SUMO1 in 293 FT cell with SUMO1-recombinant adenovirus,immunoprecipitating the cell lysates with anti-SUMO1 antibody,detecting whether p47 exists in the IP eluates using the method of Western Blot.Depletion of the potential SUMO-interaction Motif on p47 by introducing mutations at position K134 and K135,co-transfection of p47(Wt/Mutant)and mature SUMO1 plasmid DNA into 293 FT cell via lipofection,then comparing the fluorescence intensity of ROS production between Wt and Mutant-p47 transfected cells under STZ treatment;Co-transformation of p47(Wt/Mutant)and pT-E1E2S1 plasmid DNA into BL21,inducting the expression of target protein with IPTG,then conducting a GST-pulldown assay to detect whether Mutant-p47 could be sumoylated.In case p47 was not the SUMO-binding target,mice islets were isolated in batch before and after STZ treatment to examine the SUMOylation targets that related to ROS production by LC-MS/MS analysis.Results: p47 was precipitated from sumoylated proteins by method of immunoprecipitation with its band detected at approximately 100 KDa;The fluorescence intensity of STZ stimulated ROS production was a little bit stronger in Mutant-p47 transfected cells than in Wt-p47 transfected group,but the difference was not statistically significant;By method of GST-pulldown,sumoylated bands were detected in the eluation of Wt-p47 transformed bacteria protein(approximately 100 and 130 KDa),while no band was detected in Mutant-p47 transformed group,which suggested that SUMO1 could not bind to p47 with K134K135 mutation.However,no matter in Wt or Mutant-p47 transformed group,only one band at 73 KDa(presumably GST-p47)was detected in the eluation incubated with p47 antibody,it could not be confirmed that the protein at 130 KDa detected by SUMO1 antibody was sumoylated p47.Moreover,p47 was not found in the quantifiable SUMOylation target proteins by method of LC-MS/MS analysis.Conclusion: SUMOylation was proved to modulate the process of ROS generation in pancreatic ? cell under oxidative stress.However,based on the above results,we could not draw the conclusion that p47 serves as a SUMOylation target,by which SUMO1 regulates NADPH oxidase derived ROS production.The possibility that SUMO1 regulates ROS generation via interacting with other components of the NADPH oxidase could not be excluded.Besides,except for NADPH oxidase,SUMO1 may modulate the process of ROS production by interplaying with other sources of ROS derivation.Further studies are needed to address this issue.