The Role And Mechanism Of BAG3 Regulating PARP1 Ubiquitination And Degradation In Oxidative Stress-induced Vascular Endothelial Injury

Objective: Cardiovascular disease has always been one of the main threats to human health.Vascular endothelial injury is the first event of cardiovascular disease,and oxidative stress-related endothelial injury is the initiating factor of cardiovascular disease.Bcl2-related athanogene3(BAG3)is a chaperone regulator of the BAG family that interacts with various proteins and affects cell survival by activating multiple pathways.BAG3 interacts with a variety of proteins,affects cell survival by activating multiple pathways,and also plays an important role in apoptosis,cell adhesion,cytoskeleton remodeling,and autophagy.Previous evidence suggested that BAG3 is involved in the regulation of contractility and calcium homeostasis in ventricular myocytes.However,the functions and regulatory mechanisms of BAG3 in oxidative stress-related endothelial injury remain unclear.Poly(ADP-ribose)polymerase 1(PARP1)protein is a classic vascular injury protein,and one of the most important causes of endothelial injury is oxidative stress-induced hyperactivation of PARP1.Currently,PARP1 inhibitors are used in the clinical treatment of cancer;however,there are phase II evaluations showing that cardiovascular-related PARP1 inhibitors have poor clinical efficacy,suggesting that the regulatory mechanisms of PARP1 in cardiovascular disease are not fully understood.In fact,PARP1 undergoes post-translational modifications,and our previous study showed that the Nedd4 family member WWP2 is a PARP1-specific E3 ubiquitin ligase.However,the exact mechanism of the WWP2-PARP1 interaction remains unclear.This research explores the key role of BAG3 in oxidative stress-related endothelial damage,and how BAG3 exerts this role through downstream proteins and specific regulatory mechanismsMethods: Part I: Conditioned vascular endothelial-specific genotype mice and BAG3-overexpressing transgenic mice,including Tek Cre+,BAG3Fl/Fl(BAG3-e KO)and Tek Cre-,BAG3Fl/Fl(BAG3-e WT),were obtained from Shanghai model organisms,as well as BAG3-TG and BAG3-WT.BAG3 knockout or overexpression efficiency was assessed by Western Blot immunoblotting.Eight-to ten-week-old male specific pathogen-free(SPF)mice were examined and selected among all mice.In the Ang II and Na Cl infusion mouse model,BAG3-e WT,BAG3-e KO,BAG3-WT,and BAG3-TG(6mice/group)were randomly divided into models for endothelial injury and vascular remodeling induction.Mice were euthanized by decapitation after isoflurane anesthesia.The blood vessel tissue samples of mice were taken and stained with H&E and Masson’s trichrome reagent to evaluate the thickness of blood vessels and the degree of fibrosis.ROS levels in vascular endothelial cells were detected using ROS fluorescence detection method.Further verify the vascular endothelial injury at the protein level: Western blot was used to detect the protein expression levels,including: the expression of PARP-1,cleaved-Caspase3(apoptosis-related protein),Col-1,a-SMA(fibrosis-related protein),3-Nitrotyrosine(oxidative stress related protein),OGG1(oxidative stress factor)and SOD1(antioxidative stress protein).Part II: HEK293 T cells were transfected with full-length human Flag-BAG3 and subjected to co-immunoprecipitation to identify BAG3-interacting proteins by mass spectrometry to obtain gene ontology,biological processes,molecular functions,and cellular component enrichment analysis of BAG3-interacting proteins.The PARP1 mass spectrum was extracted,and co-immunoprecipitation biological experiments were performed to verify the interaction between BAG3 and PARP1.The interaction between BAG3 and PARP1 was verified by using an anti-BAG3 antibody,and the interaction between PARP1 and BAG3 was verified by using an anti-PARP1 antibody,and it was determined that the interaction between BAG3 and PARP1 was maintained in the presence of Ang II.In addition,co-immunoprecipitation experiments were conducted between BAG3 and each truncated domain of PARP1 to further explore which domain of PARP1 Zinc1-Zinc2 domain,BRCT domain,PARPA helix domain and PARP catalytic domain interacts with BAG3.Part III: In human umbilical vein endothelial cells(HUVEC),gradient overexpression experiments were performed by transfecting Flag-BAG3 plasmid,and a stable knockdown cell line of lentiviral transfection of sh BAG3 was constructed to explore its effect on endogenous PARP1 levels.HUVECs were divided into two groups and four groups,namely Control group and Flag-BAG3 overexpression group,NC group and sh BAG3 knockdown group.After treatment with cycloheximide(CHX)and proteasome inhibitor Mg132 for different time,the expression level of PARP1 was evaluated by Western Blot with time,to determine whether BAG3 regulates PARP1 through the transcriptional pathway or the proteasomal degradation pathway.The effect of exogenous expression of BAG3 on the ubiquitination of PARP1 was verified byco-immunoprecipitation.Next,the changes in the interaction level between WWP2 and PARP1 were verified by co-immunoprecipitation experiments in HUVEC cells overexpressing Flag-BAG3 and sh BAG3 knockdown cell lines to explore the level of PARP1 ubiquitination by WWP2 in sh BAG3 cells and in cells overexpressing exogenous BAG3.By comparing the ubiquitination levels of PARP1-WT and specific point mutant PARP1,the specific PARP1 site at which BAG3 induced the ubiquitination of PARP1was determined.Results: Part I: 1.BAG3 endothelial-specific knockout mice displayed significantly aggravated Ang II-related endothelial injury and post-injury remodeling,including vascular thickening,vascular fibrosis and angio-ROS generation,vascular injury proteins PARP1 and Cleaved-Caspase3 levels were significantly increased;vascular fibrosis proteins Col-1 and a-SMA levels were significantly increased;oxidative stress factors3-Nitrotyrosine and OGG1 were significantly increased,while Ang II was associated with anti-oxidative stress The amount of the protein SOD1 was significantly reduced.2.BAG3 transgenic mice exhibited significant alleviation of Ang II-induced vascular endothelial injury and post-injury remodeling: BAG3 transgenic(BAG3-TG)mice were significantly reduced in Ang II compared with BAG3-WT mice.Vascular thickening,vascular fibrosis,and vascular ROS generation were significantly attenuated after induced vascular endothelial injury.Compared with BAG3-WT mice,BAG3-TG mice significantly reduced the levels of Ang II-induced vascular injury factors PARP1 and Cleaved-Caspase3;compared with BAG3-WT mice,Ang II in BAG3-TG mice The induced vascular fibrosis proteins Col-1 and a-SMA levels were significantly reduced;BAG3-TG mice had significantly lower levels of 3-Nitrotyrosine and OGG1 and significantly higher SOD1 levels compared with BAG3-WT mice.Part II: BAG3 interacting proteins were identified based on mass spectrometry,gene ontology,biological process,molecular function and cellular component enrichment analysis showed that the oxidative stress damage protein PARP1 may be a new key potential interacting protein of BAG3 and the spectrum was extracted.Next,endogenous immunoprecipitation was demonstrated by co-immunoprecipitation experiments using anti-BAG3 antibody and anti-PARP1 antibody to verify the interaction of BAG3 with PARP1.At the same time,we determined that the interaction between BAG3 and PARP1 was maintained in the presence of Ang II.Not only that,co-immunoprecipitation results showed that BAG3 binds to the BRCT domain of PARP1(PARP1-BRCT).Part III: 1.BAG3 promotes the proteasomal pathway degradation of PARP1 through ubiquitination modification: the increase of BAG3 overexpression gradient is associated with the gradual decrease of endogenous PARP1 level,and the endogenous PARP1expression level is increased after BAG3 knockdown in HUVEC cells.NC cells showed a significantly higher PARP1 degradation rate(slope)after CHX treatment,while the sh BAG3 group maintained a low degradation rate of PARP1 over time;compared with sh BAG3 cells,after administration of MG132 in NC cells,PARP1 Protein accumulation was faster,and the final level of PARP1 in the two cells was roughly the same;compared with the control group,the rate and slope of PARP1 protein accumulation in the BAG3overexpression group were lower after MG132 treatment,and the final level of PARP1 in the two cells was roughly the same;compared with the control group In contrast,the degradation rate(slope)of PARP1 was higher in the BAG3 overexpression group after CHX treatment.It was next confirmed that exogenous overexpression of BAG3 promotes the ubiquitination of PARP1,which is consistent with the degradation of PARP1 through the proteasomal pathway.2.BAG3 enhances the ubiquitination of PARP1 by WWP2,and the modification site is K249;the interaction between the E3 ubiquitination ligase WWP2 and PARP1 in cells transfected with Flag-BAG3 compared with control cells levels increased 2.079-fold(***,P<0.001).Consistent with the above results,the level of PARP1 ubiquitination of WWP2 was lower in sh BAG3 cells and higher in cells overexpressing exogenous BAG3.We found that BAG3 enhanced the ubiquitination level of PARP1-WT but not PARP1-K249 R,suggesting that BAG3 promotes ubiquitination and degradation of PARP1 by enhancing the ubiquitination modification of K249 by WWP2.Conclusion: 1.In mouse vascular endothelium,decreased BAG3 expression leads to significantly aggravated oxidative stress-related vascular endothelial injury and post-injury remodeling,whereas increased BAG3 expression leads to significantly alleviated oxidative stress-related vascular endothelial injury and remodeling after injury.2.PARP1 is a downstream binding protein of BAG3,and BAG3 interacts with the BRCT domain of PARP1,and the interaction is enhanced under the action of Ang II.3.BAG3enhances the interaction between the E3 ubiquitination ligase WWP2 and PARP1,and enhances the level of ubiquitination and degradation of PARP1 by modifying the K249ubiquitination site,thereby reducing the expression level of PARP1 in cells,which is beneficial to the protection against oxidative stress-induced vascular endothelial injury.