Study On Regulation Effect Of NF-κB By S-nitrosylation

This study aimed to examin the inactivation of NF-κB which controls the espreesion of many genes invovled in the inflammatory response, and the negative regulation of NF-κB activating pathways by S-nitrosylation.In the present study, incubation with 300μM of nitrosating agent SNP for 8h can create a NO sufficient microenvironment to induce the occurrence of protein S-nitrosylation, while have little cytotoxic effect to PC 12 cells. Technique combination of biotin switch assay and western blot successfully detected that treatment of NO donor SNP resulted in a large number of S-nitrosylated proteins.293T cells transiently transfected with plasmid pcDNA6-NF-KB p65, overexpressed p65, one of the most common subunit of NF-κB. After incubation with 300μM of SNP for 8h, S-nitrosylated p65 in the whole cell was detected using the technique combination of the biotin switch assay, protein purification and western blot. And the luciferase reporter assay showed that S-nitrosylation of p65 markedly suppressed the transcriptional activity of NF-κB.The results suggest that NO has the ability to result in a large number of S-nitrosylated proteins, among which, S-nitrosylation of p65 provides a new mechanism for inhibition of the transcriptional activity of NF-κB, inactivation of NF-κB signaling pathway and attenuation of inflammatiory reaponse.