Study On The Effects Of Cathepsin B S-nitrosylation Modification On Angiotensin Ⅱ-induced Endothelial Injury And Its Mechanism

Cathepsin B(CTSB),a member of the lysosomal cysteine protease family,is involved in regulating various cellular biological functions,such as innate immune response,extracellular matrix degradation,inflammatory response and cell apoptosis.CTSB is associated with a variety of pathological and carcinogenic processes.The Snitrosylation of protein,a kind of protein translation modification,is formed by the reaction of the free sulfhydryl group(-SH)within protein cysteine residues and nitric oxide(NO).The protein S-nitrosylation has been found to be involved in several signaling pathways.In this study,NO donor sodium nitroprusside(SNP)was used to treat vascular endothelial cells,then proteins were extracted from endothelial cells.The S-nitrosylated proteins were detected by using biotin-switch and LC-MS/MS assays.Furthermore,by site-directed mutagenesis and biotin-switch assay we identified that CTSB was S-nitrosylated at Cys319 site in SNP-treated endothelial cells.Additionally,angiotensin II(Ang II)also induced S-nitrosylation of CTSB at Cys319 site in endothelial cells.Furthermore,Ang II promoted the up-regulation of iNOS in endothelial cells,and inhibition of iNOS with 1400 W could inhibit Ang II-induced CTSB S-nitrosylation.The S-nitrosylation of CTSB increased Ang II-induced NLRP3 inflammasome activation and cell senescence of endothelial cells.Surprisingly,we found that the 3’ untranslated region(3’ UTR)of CTSB mRNA contains Alu element(2189-2472 bp)and the A-to-I RNA editing frequency of CTSB mRNA was significantly increased in Ang II-treated endothelial cells.The inhibition of both iNOS and CTSB S-nitrosylation reduced Ang II-induced A-to-I RNA editing of CTSB mRNA.Moreover,Ang II increased the expression of ADAR1 protein in endothelial cells,while suppression of both iNOS and CTSB S-nitrosylation diminished Ang IIinduced ADAR1 protein overexpression.Additionally,ADAR1 was exported from the nucleus and translocated to the cytoplasm in Ang II-treated endothelial cells.Importantly,we found that both ADAR1 protein level and A-to-I RNA editing frequency of CTSB mRNA were increased in the aortic vascular tissue of aortic dissection patients compared with healthy controls.Therefore,the present study revealed that Ang II induced the S-nitrosylation of CTSB protein at Cys319 site via upregulating iNOS,thus promoting Ang II-induced NLRP3 inflammasome activation and cell senescence in vascular endothelial cells.Moreover,Ang II also increased the expression of ADAR1 protein and the A-to-I RNA editing frequency of Alu elements in CTSB mRNA 3’ UTR and might be partly dependent on iNOS activation and CTSB S-nitrosylation.