Regulation Of SIRT2 On The Expression Of Inflammatory Factors By Hsp90-GR Signal

BACKGROUND:SIRT2(Silent information regulator 2)is an NAD +-dependent deacetylase that is an important member of the Sirtuins family.SIRT2 is not only mainly present in the cytoplasm,but can be translocated into the nucleus to exert its related functional effects.Recent studies have shown that SIRT2 interacts with a variety of cellular processes including energy metabolism,cell proliferation,cell differentiation,apoptotic cell aging and metabolism,genomic stability,inflammation,longevity,and tumorigenesis.In the past few years,many SIRT2 protein substrates have been identified,and SIRT2 can deacetylate multiple proteins and participate in many cellular processes,including regulation of inflammation.However,its exact role in the inflammatory process has not been fully elucidated.Our previous study found a new substrate protein heat shock protein(Heat Shock Protein 90,Hsp90)that interacts with SIRT2.Based on this,we studied and explored the interaction between SIRT2 and Hsp90 and explored the pair.Its regulatory mechanism of downstream inflammatory factor expression.It provides a new theoretical basis and treatment ideas for the future development of immune response targeting SIRT2 regulation and inflammation-related diseases.PURPOSE:Based on the identification of the novel substrate protein Hsp90 of SIRT2 in the early stage,we further confirmed the mutual regulation relationship between SIRT2 and heat shock protein Hsp90,and then analyzed the SIRT2 pair by using the cell line B104 stably overexpressed or silenced by SIRT2 in the laboratory.The regulation of Hsp90 and its downstream factors.The first is to detect the effect of SIRT2 on the acetylation level of Hsp90 protein.The second is to detect the effect of SIRT2 on the interaction between Hsp90 and glucocorticoid receptor(GR);and then further to detect the effect of SIRT2 overexpression on GR subcellular localization.The effect of its transcriptional activity;finally,through a series of experiments to detect the regulation of SIRT2 on the expression level of inflammatory factors under Hsp90,to further analyze the cellular biological function of its action.METHODS:First,we used the GST-pulldown assay and the co-IP assay to further validate the interaction between SIRT2 and the heat shock protein Hsp90.Cell regeneration and culture were then performed on cell lines that had been screened for stable SIRT2 overexpression or silencing,and then cells were harvested for protein extraction and co-IP experiments were performed to examine whether SIRT2 and Hsp90 proteins were bound together.The experiment was then used to examine the effect of SIRT2 on the interaction between Hsp90 and the glucocorticoid receptor GR.The effect of SIRT2 overexpression on GR subcellular localization was then examined by nuclear separation assay.At the same time,the luciferase reporter gene assay was used to examine the effect of SIRT2 overexpression on the transcriptional activity of the glucocorticoid receptor GR.Finally,we examined the effects of SIRT2 on the mRNA and protein levels of downstream inflammatory factors of Hsp90 by qPCR and Western blot,respectively.Hsp90 mutations were generated by PCR,and the acetylated and non-acetylated mutant plasmids were respectively transfected into Jurkat cells.Then,after confirming that both mutants were overexpressed,Western blot was used to further verify whether acetylation of Hsp90 K294 plays an important role in mediating the expression of inflammatory factors,with or without lipopolysaccharide LPS stimulation.The role of analyzing possible signaling pathways.RESULTS:1.We further verified the interaction between SIRT2 and heat shock protein Hsp90 by GST-pulldown test and co-IP experiment,and found that the band that binds SIRT2 to Hsp90 can be detected,and stimulated without lipopolysaccharide LPS.In contrast,in B104 cells stimulated by LPS,the binding between SIRT2 and Hsp90 appeared to be stronger.Moreover,bands in which SIRT2 binds to Hsp90 can also be detected in B104 cells.2.Co-IP experiments in the study of the functional relationship between SIRT2 and Hsp90 showed that although the total protein level of Hsp90 was comparable in SIRT2 overexpression and control cells,and SIRT2 overexpression did not cause changes in Hsp90 total protein levels,However,the level of Hsp90 acetylation showed a decreasing trend after SIRT2 overexpression.In contrast,the level of Hsp90 acetylation in SIRT2-silenced B104 cells showed an increasing trend compared to the total Hsp90 expression level,indicating that SIRT2 has deacetylation of Hsp90 in B104 cells.3.Next we used the co-IP assay to examine the effect of SIRT2 on the interaction between Hsp90 and the glucocorticoid receptor GR.We performed an IP assay using an antibody against Hsp90,and found that the binding amount of the glucocorticoid receptor GR to Hsp90 was decreased in SIRT2 overexpressing B104 cells compared with the control group.In contrast,the binding of the glucocorticoid receptor GR to Hsp90 was increased in SIRT2-silenced B104 cells.We performed IP assays using anti-GR antibodies and found similar IP measurements with Hsp90 antibodies.The binding amount of GR to Hsp90 in B104 cells overexpressing SIRT2 was reduced compared to the control group with or without lipopolysaccharide LPS stimulation.In contrast,the binding of the glucocorticoid receptor GR to Hsp90 was increased in SIRT2-silenced B104 cells.More interestingly,we measured with an anti-acetylated Hsp90 antibody,and the level of interaction of Hsp90 with GR in SIRT2 overexpressed or silenced B104 cells corresponds to the effect of SIRT2 overexpression or silencing on Hsp90 acetylation levels.These results demonstrate SIRT2 De-cleavage of GR and Hsp90 is facilitated by deacetylation of Hsp90.4.We then found through nuclear separation experiments that in SIRT2 overexpressing B104 cells,the protein level of GR in the nucleus was higher than that in the cytoplasm.In contrast,in SIRT2-silenced B104 cells,the protein level of GR in the nucleus was lower than that in the cytoplasm.These results indicate that Sitr2 overexpression alters the ratio of nuclear to cytoplasmic GR,indicating that SIRT2 is deacetylated by Hsp90,which induces its dissociation from GR,which ultimately leads to GR translocation to the nucleus.5.We also measured the effect of SIRT2 on the binding ability of GR in B104 cells to its Glucocorticoid receptor response element(GRE)by luciferase reporter gene assay.Western blot was first used to verify that SIRT2 was indeed overexpressed or silenced in B104 cells to further confirm the reliability of the luciferase reporter gene assay results.Then,based on this,the luciferase reporter gene was tested and the results showed that SIRT2 overexpression enhanced the expression of the luciferase reporter gene compared to the control cells.In contrast,SIRT2 silencing attenuated the expression of the luciferase reporter gene,indicating that SIRT2 is effective in increasing the transcriptional activity of GR.6.Next,we examined the expression of SIRT2 and cytokines TNF?,IL-1 and IL-6 in six cell lines by Western.It was found that Jurkat cells were able to simultaneously express SIRT2 and the above three cytokines in these six cell lines.Therefore,we used the cells to detect the effect of SIRT2 on the expression of inflammatory factors downstream of Hsp90 by qPC and Western blot.The results showed that SIRT2 overexpression inhibited the expression of inflammatory cytokines at both mRNA and protein levels,stimulated with or without lipopolysaccharide LPS,while SIRT2 silencing promoted inflammatory cytokines.expression.It was also found that SIRT2 overexpression significantly inhibited the expression of inflammatory cytokines compared to SIRT2 silencing under the stimulation of lipopolysaccharide LPS.7.Finally,we play an important role in determining whether acetylation of Hsp90 K294 mediates the expression of inflammatory factors.We constructed a mutant of acetylated(K294Q)and Hsp90 non-acetylated(K294R)of Hsp90.Western analysis of Jurkat cells showed that both mutants of Hsp90 were overexpressed.These two mutants were then overexpressed in SIRT2 overexpressed or silenced Jurkat cells,and were subjected to Western blot analysis with or without lipopolysaccharide LPS,respectively.It was found that Hsp90 acetylation mimetic mutations were associated with Hsp90 stimulated with or without LPS.The expression levels of these three inflammatory factors were increased compared to the non-acetylation mutation.It was also found that the Hsp90 mimetic acetylation(K294Q)mutant almost eliminated the effect of SIRT2 overexpression or silencing in Jurkat cells compared to wild-type Hsp90,and more interestingly,compared to the control,lipopolysaccharide LPS Stimulation appears to have a greater effect on the expression of inflammatory cytokines in Hsp90 mimic mutant(K294Q)Jurkat cells.These results fully demonstrate the inhibitory effect of SIRT2 in the inflammatory response.CONCLUSION:SIRT2 and Hsp90 can bind in vitro and in vivo and deacetylate Hsp90,promote the dissociation of glucocorticoid receptor GR and Hsp90,and finally cause GR to translocate into the nucleus to promote the binding of GR to GRE to inhibit inflammatory cytokines.expression..