The E3 Ubiquitin Ligase Smurf2 Binds And Regulates Stability Of PARP1 To Inhibit Apoptosis Of Human Umbilical Vein Endothelial Cells Under Oxidative Stress

Objective:Oxidative stress injury plays a very important role in many cardiovascular diseases.Oxidative stress is involved in the pathophysiological process of a series of cardiovascular diseases,such as atherosclerosis,myocardial ischemia-reperfusion injury,hypertension,heart failure,and so on.It is also a hot spot in the study of cardiovascular diseases.Ubiquitination is a ubiquitous post-translational modification of proteins that controls a wide range of biological functions and plays a crucial role in maintaining the homeostasis of cells in physiology and disease.Many studies have shown that oxidative stress damage is inextricably linked to ubiquitination.Methods:1.HUVEC was selected as the experimental object in this study.HUVEC was cultured and stimulated by H2O2 to induce oxidative stress injury of endothelial cells.Cell viability was observed by CCK-8,apoptosis level was detected by flow cytometry,ROS active oxygen kit,Smurf2 and related apoptosis protein were detected by West bolt.2.Flag-Smurf2 was transfected into cells by plasmid transfection,and H₂O₂ was stimulated at the same time.Cell viability was observed between groups by CCK-8.Flow cytometry,ROS reactive oxygen kit was used to detect apoptosis.,West Bolt detects expression of Smurf2 and related apoptotic proteins.By knocking down Smurf2 and simultaneously stimulating H₂O₂,the cell viability was observed between groups by CCK-8,the level of apoptosis was detected by flow cytometry,ROS reactive oxygen kit,and the expression of Smurf2 and related apoptosis proteins was detected by West Bolt.Transfection of Flag-Smurf2 and Myc-PARP1 simultaneously gave H₂O₂ stimulation.Flow cytometry was used to detect the level of apoptosis.West Bolt was used to detect the expression of related apoptotic proteins.Transfection of Flag-Smurf2,simultaneous stimulation with H₂O₂ and Heclin,detection of apoptotic levels by flow cytometry,and expression of related apoptotic proteins by West Bolt.3.Co-IP experiment to observe the binding of Smurf2 to PARP1 and its binding region.4.Overexpression and knockdown of Smurf2 and detection of PARP1 expression using Wset-Blot.5.Construction of Smurf2 stable knockdown HUVEC cell line and construction of Smurf2 functional site-inactivated Smurf2?C716G?plasmid.Flag-Smurf2 and Flag-Smurf2?C716G?were over-expressed in Smurf2 knockdown cells.PARP1expression was detected by West Bolt.The co-IP experiment was used to observe the interaction between Smurf2?C716G?and PARP1.West Bolt was administered to CHX in cells overexpressing and knocking down Smurf2 to detect half-life changes in PARP1,and MG132 was overexpressing and knockdown in cells to detect PARP1accumulation using West Bolt.6.Co-immunoprecipitation experiments were performed to observe the interaction between Smurf2 and PARP1 under MG132 stimulation.Heclin was also administered to Flag-Smurf2 cells overexpressing PARP1 expression using West Bolt.Co-immunoprecipitation experiments to observe overexpression and knock down Smurf2 to observe Smurf2 ubiquitination of PARP1.Results:1.The apoptosis level,ROS level,Smurf2 expression and apoptosis related proteins cleaved PARP1 and cleaved caspase 3 expression of HUVEC increased with the aggravation of oxidative stress,meanwhile,the cell viability of HUVEC decreased.2.A Flag-Smurf2 plasmid and then stimulated with H2O2.We found that the overexpression of Smurf2 significantly reduced the expression of cleaved PARP1 and cleaved caspase3 and decreased HUVEC apoptosis.A HUVEC viability assay and flow cytometry followingannexin-FITC/PI staining and ROS assy showed that the overexpression of Smurf2 alleviated apoptosis.We then identified the most efficacious Smurf2 siRNA fragment from three fragments.We transfected HUVECs with control siRNA and siRNA fragment 3 and then stimulated the HUVECs with H₂O₂.The expression of cleaved PARP1 and cleaved caspase3 in Smurf2 knockdown cells was significantly higher than that in the negative control group,and HUVEC injury was aggravated.The results of a HUVEC viability assay and flow cytometry after annexin-FITC/Pi staining,ROS assy also showed that Smurf2 knockdown increased apoptosis.We overexpressed Myc-PARP1 and Flag-Smurf2 together in HUVEC,we have observed that overexpression of Myc-PARP1 can aggravate oxidative stress injury of H₂O₂,overexpression of Flag-Smurf2 on the basis of overexpression of Myc-PARP1 can rescue the damage to some extent.At the same time,flow cytomet followingannexin-FITC/PI staining results were the same as before.We overexpressed Flag-Smurf2 and give Smurf2 inhibitor Heclin in HUVEC.Under the stimulation of oxidative stress,we confirmed that Smurf2 lost its ability to protect HUVEC from oxidative stress injury and Flow cytometry following annexin-FITC/PI staining results also confirm this conclusion3.We confirmed by endogenous coimmunoprecipitation that Smurf2 is a novel protein that interacts with PARP1 and that Smurf2 and PARP1 enhance each other after stimulation with H₂O₂.At the same time,we determine the combination area between Smurf2 and PARP1.4.The overexpression of Flag-Smurf2 affected the expression of PARP1,which indicates that Smurf2 can regulate the stability of PARP1.The expression of PARP1was significantly upregulated in cells transfected with three siRNA fragments against Smurf2.Similar results were observed in a cell line in which Smurf2 had been stably silenced.Then,we found that both endogenous and exogenous PARP1 expression depended on and were negatively correlated with the expression of Flag-Smurf2.5.We overexpressed Flag-Smurf2 and Flag-Smurf2?C716G?in cells in which Smurf2had been stably knocked down.The expression of endogenous PARP1 in Smurf2mutant cells was higher than that in cells transfected with Flag-Smurf2.Furthermore,the interaction between Smurf2?C716G?and PARP1 was weakened,as shown by coimmunoprecipitation experiments.We overexpressed Flag-Smurf2 and treated the resulting cells with cycloheximide?CHX?;the half-life of endogenous PARP1 in these CHX-treated cells was shorter than that in the empty vector group.Next,we treated Smurf2 knockdown cells with CHX and found that the knockdown of Smurf2increased the shortened half-life of endogenous PARP1.The same experimental results were obtained in CHX-treated cells overexpressing Myc-PARP1 and Flag-Smurf2.We transfected cells with empty vector Flag-Smurf2 and then administered the proteasome pathway inhibitor MG132.We found that MG132 could block the Smurf2-mediated negative regulation of PARP1 and that endogenous PARP1accumulation was more pronounced.This accumulation of PARP1 was further amplified when MG132 was administered to Smurf2 knockdown cells.6.We transfected cells with empty vector or Flag-Smurf2 and administered MG132and Heclin restored the expression of PARP1.In addition,the interaction between Smurf2 and PARP1 was enhanced by MG132.We therefore transfected cells with Flag-Smurf2 and Flag-Smurf2?C716G?and used HA-UB for coimmunoprecipitation experiments.Smurf2 increased the level of ubiquitinated PARP1.Similarly,Smurf2silencing reduced the level of ubiquitinated PARP1 in Smurf2 knockdown cells).Further,we transfected Flag-Smurf2,HA-UB and gave Smurf2 inhibitors Heclin for coimmunoprecipitation experiments.Heclin inhibited the ubiquitination of PARP1.Conclusion:The results showed that Smurf2,as an E3 ubiquitin ligase,participated in the apoptosis of HUVEC induced by oxidative stress,thus alleviating the ROS production and HUVEC apoptosis induced by H2O2.At the same time,we found that Smurf2 can combine with PARP1 and degrade PARP1 through ubiquitin proteasome pathway.Under oxidative stress,Smurf2 can protect HUVEC and alleviate oxidative stress injury by degrading and activating PARP1 through ubiquitin proteasome pathway.