Mechanism Of DNA Base Damage Repair Enzyme OGG1 Regulating Phosphorylation Of RelA/p65 Ser276

Organisms have always been in the environment of reactive oxygen species(ROS)produced by external environmental factors and their own metabolic activities.When the content of reactive oxygen species in cells exceeds their own clearance rate,oxidative stress will form,causing proteins,lipids and DNA Damage,but damage to DNA as genetic material is the most deadly.Guanine has the lowest redox potential among the four bases of DNA,and is most easily oxidized to 8-oxo G.The damage can be repaired through the base excision repair pathway initiated by OGG1.Although guanine is easily oxidized,72% persent of the genes in the vertebrate promoter region are GC-rich.In recent years,more and more evidence has shown that 8-oxo G is not only an oxidative damage,but also an epigenetic modification.By binding to its specific repair protein OGG1,it recruits NF-?B to its binding site.Realize the transcriptional regulation of inflammatory genes.NF-?B is the main regulate of pro-inflammatory gene expression and is very important for maintaining the homeostasis of the immune system.The NF-?B family includes five subunits,p50,p52,RelA/p65,Rel B and c-Rel,and most of them exist in the form of p65-p50 heterodimers in cells.The classical activation pathway of NF-?B believes that the nucleus of p65-p50 dimer is very important for NF-?B transcriptional activation,but studies have found that the more important thing is the RelA/p65 phosphorylation modification that occurs in the nucleus.RelA/p65 has many phosphorylation sites(S276,S529,S536),but the most concerned is the phosphorylation of RelA/p65 S276,which has the significance of changing the conformation of RelA/p65 and enhancing the transcriptional activity of NF-?B.Preliminary work in the laboratory has shown that under inflammatory stimulation,OGG1 has no effect on the nucleus entry of RelA/p65,but regulates the phosphorylation of RelA/p65 S276 and promotes the expression of Cxcl2 and IL8 genes,but the expression of I?B? genes is not affected(with ROS sensitive NF-?B transcription regulation results are consistent).Since OGG1 is only a DNA damage repair enzyme and does not have protein kinase activity,it cannot directly regulate the phosphorylation of RelA/p65 S276.At the same time,looking up the literature,we found that there are 6 kinds of kinases MSK1,MSK2,PKAc,PKC,RSK p90,Pim1 that can regulate the phosphorylation of RelA/p65 S276.Therefore,it is speculated that OGG1 may regulate the phosphorylation of RelA/p65 S276 by recruiting kinases under inflammatory stimulation.This experiment firstly used TNF? to stimulate HEK293 cells to establish an inflammatory stimulus model;secondly,it verified that when OGG1 regulates the phosphorylation of RelA/p65 S276 under inflammatory stimulation,RelA/p65 interacts with OGG1;finally,under inflammatory stimulation,OGG1,RelA/p65,and MSK1(Mitogen and stress-activated protein kinase 1)are in the same complex.It was verified that the kinases MSK1 and PKAc can regulate the phosphorylation of ROS-sensitive RelA/p65 S276,and in the in vitro GST-Pull Down experiment,OGG1 and MSK1 have a direct interaction.OGG1 can bind to the N-terminal kinase trans domain(NTKD)of MSK1.Under inflammatory stimulation,the OGG1 inhibitor TH5487 attenuates the interaction between MSK1 and RelA/p65,thereby regulating the phosphorylation of RelA/p65 S276.These experimental results reveal OGG1 regulates the mechanism of RelA/p65 S276 phosphorylation modification by recruiting MSK1.In short,under inflammatory stimulation,OGG1 recruits MSK1 to regulate the phosphorylation of RelA/p65 S276 and regulate the expression of some genes in the NF-?B range.Preliminary revealed the new mechanism of OGG1 involved in gene transcription activation,and further analyzed the mechanism that 8-oxo G and OGG1 specifically direct the rapid activation of the transcriptome in response to oxidative stress.In vitro GST-Pull Down experiment proved that OGG1 can directly bind to NTKD of MSK1.This provides a basis for the design of small molecule inhibitors to specifically block the expression of pro-inflammatory genes and improve the treatment strategies of excessive inflammation and chronic inflammation-related diseases caused by innate immune diseases,which have important medical significance.