Research On PRMT5 Methylation Of TRAF2 And Nur77 And Its Related Mechanism

PRMT5 is a type II arginine methyltransferase that can induce symmetric arginine methylation.Arginine residues of histone and non-histone substrates can be methylated.Research shows that it plays an important role in the regulation of gene transcription.PRMT5 is known to be involved in multiple protein complexes.Therefore,it is necessary to study the methylation function of PRMT5.We explored the methylation of PRMT5 on tumor necrosis factor receptor-related factor TRAF2 and nuclear orphan receptor Nur77 from the following perspectives,and further explored the mechanism.First,we used techniques such as co-immunoprecipitation,GST-Pulldown,and immunofluorescence to explore the interaction between PRMT5 and TRAF2,and we constructed different TRAF2 fragment plasmids to lock the interaction in the TRAF domain of TRAF2.To further study the methylation effect of PRMT5 on TRAF2,we transfected wild type(WT)and domain-negtive PRMT5(DN-PRMT5)in 293 T cells,and found that WT-PRMT5 can methylate TRAF2.At the same time,we detected that PRMT5 regulates TRAF2 protein expression and further promotes TRAF2 ubiquitination to a certain extent.TRAF2 is a key adaptor protein in the NF-?B signaling pathway,therefore,the effect of PRMT5 on NF-?B signaling pathway was evaluated by Western blot.Interestingly,we found that the expression of NF-?B kinase NIK was significantly increased in TRAF2 overexpressed cell,however,this increase in NIK was suppressed after co-transfected with PRMT5 plasmid.These datasuggest that PRMT5 may affect the non-classical NF-?B signaling pathway.Then,we treated the cells in the same way and examined the changes of P100 / P52,and confirmed that TRAF2 would promote the accumulation of P52 and this accumulation would be inhibited after overexpressing PRMT5.The ubiquitination of NIK plays a key role in the non-classical NF-kB signaling pathway.At present,there are two hypotheses about the mechanism of the ubiquitination of NIK:(1)TRAF3 and NIK directly interact to degrade NIK by ubiquitination;(2)TRAF2 / TRAF3 acts as a bridge to promote cIAPs to ubiquitinate NIK.Either way is inseparable from the role of TRAF3,so we tested the ubiquitination of TRAF3 and NIK.Next,we further explore the interaction of PRMT5 on cIAP1 and TRAF2 and the effect of cIAP on the ubiquitination of TRAF2,providing a theoretical basis for further explaining the role of PRMT5 on TRAF2-mediated NF-?B signaling pathway.Second,we studied the methylation effect of PRMT5 on Nur77.First,we detected that PRMT5 directly interacts with Nur77,and Nur77 is a methylated substrate of PRMT5.Secondly,we found that the flavonoid dihydromyricetin(DHM)previously studied by our research group can inhibit the interaction between PRMT5 and Nur77,and DHM can promote Nur77 expression and AMPK phosphorylation in a dose-dependent and time-dependent manner.In order to further clarify the relationship between Nur77 and DHM,we confirmed the physical binding of Nur77 's ligand binding domain(LBD)and DHM by molecular docking,circular dichroism chromatography and isothermal titration calorimetry,indicating that LBD does indeed exist with DHM interaction.We further constructed a type 2 diabetes model on C57 mice,and intragastrically administered DHM after the blood glucose stabilized.It was found that DHM can treat type 2 diabetes and the therapeutic effect is comparable to the positive drug metformin.The results show that DHM can significantly improve the glucose tolerance of diabetic mice.In order to further determine whether the role of DHM in treating diabetes is related to Nur77,we also constructed a type 2 diabetes model on Nur77 gene knockout mice.After intragastric administration of DHM,itwas found that DHM did not have a significant therapeutic effect on Nur77 gene knockout diabetic mice.It is suggested that DHM treatment of diabetes may be related to Nur77.In conclusion,this paper mainly discusses the PRMT5 methylated TRAF2 to regulate the non-classical NF-?B signaling pathway;clarifies that DHM may be involved in the regulation of PRMT5 and Nur77 complex,and DHM treatment of type 2 diabetes may rely on the nuclear orphan receptor Nur77,for Provide theoretical basis for the development of diabetes treatment drugs.