The Regulation Mechanisms Of E3 Ubiquitin Ligases In Wnt And NF-?B Signaling

Ubiquitination is the process by which ubiquitin covalently binds to the targeted proteins.In this process,E3 ubiquitin ligases specifically assist ubiquitins in attaching to the target proteins,which further regulate the degradation of target proteins and the transduction of signaling pathways.The E3 ubiquitin ligases are increasingly concerned due to the important role of ubiquitination in various life activities and its specific recognition capability,while the function of E3 ubiquitin ligases in the transduction of signaling pathways are still under investigation.This study took the E3 ubiquitin ligases carboxy-terminus of Hsp70-interacting protein(CHIP)and tripartite motif-containing 25(TRIM25)as the research objects,and deeply explored the important role of E3 ligases in signaling transductions.CHIP is a U-box E3 ligase which plays important role in cytoplasmic molecular chaperone system,participats in the regulation of various signaling pathways,and also is related to multiple neurodegenerative diseases.It has been reported that the mutant of CHIP(p.T246M)causes the occurrence of Spinocerebellar autosomal recessive 16(SCAR16).SCAR16 is a neurodegenerative disease which is characterized by cerebellar atrophy and ataxia.Although this disease has been known for more than 100 years,the therapeutic methods are limited in clinical.Therefore,it's of great significance for studying the pathogenic mechanisms of SCAR16 caused by CHIP mutant when we elucidate the regulation of CHIP in the related signaling pathways.It also lays the foundation for the treatment of this disease.The TRIM family is a protein family with the most members of E3 ligases which contains RING-finger domain,B box domain,and coiled-coil domain.It is involved in various physiological processes,including cell proliferation,immunity,and antiviral response.It has been reported that many TRIM family members participate in the regulation of TNF?-induced NF-?B signaling which involve in the occurrence of various diseases such as inflammations,autoimmune diseases and tumors.However,it's unclear whether TRIM25,as a member of TRIM family,is involved in the regulation of this pathway.Furthermore the molecular mechanisms of TRIM25 in TNF?-induced NF-?B signaling still need to be studied.Based on the above findings,we investigated the signaling pathways which CHIP affected and their regulation mechanisms,and explored the regulatory effects and molecular mechanisms of TRIM25 in TNF?-induced NF-?B signaling using bioinformatics methods and biological experiments.This study was divided into two parts correspondingly.In the first part,we firstly analyzed the data obtained from the hybridization of protein microarrays using bioinformatics analysis,and screened the interacting proteins of CHIP or CHIP TPR-CC detecting the region of enzyme activity.262 and 167 proteins were respectively identified as CHIP or CHIP TPR-CC interacting proteins in the obtained two datasets of positive candidate proteins.We found many candidate proteins were enriched in cytoplasm and nuclei,and extensively participated in the process of signal transduction when we analyzed the datasets of candidate proteins with Gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis.We found ?-catenin was located in the key position of interaction network analyzed by protein-protein interaction network(PPI)network.Thus we specifically explored the regulation mechanism of CHIP in the transduction of Wnt signaling.Overexpression of CHIP promoted the activation of Wnt signaling in HEK293T cells,while the inactive mutant CHIP(p.T246M)lost its function detected by immunoblotting and real-time PCR.Knockdown or knockout of CHIP inhibited the transduction of Wnt signaling,as well as the proliferation and migration of cells in SHSY5Y or HT22 cells.We also found Wnt signaling was inhibited in the diseased SCAR 16 rats with CHIP(T246M)mutant.CHIP enhanced nucleus accumulation of ?-catenin detected by cytosolic and nuclear separation experiment and fluorescence analysis.It showed that CHIP could promote the activation of Wnt signaling.Then we explored the molecular mechanisms of CHIP in the regulation of Wnt signaling.CHIP had interaction with LEF1 screening by ZDOCK server,immunoblotting,and immunoprecipitation.We further found the E3 ligase CHIP promoted the K63-linked ubiquitination of LEF1,and accelerated the interaction between ?-catenin and LEF1 by immunoprecipitation.While the inactive mutant CHIP(p.T246M)didn't have the function,and knockdown of CHIP had the opposite function.These results indicated that CHIP had an important regulation on the transduction of Wnt signaling.The mutant of CHIP resulted in the long-term inhibition of Wnt signaling which might be the cause of SCAR16.In the second part,we screened two useful datasets GSE142986 and GSE40838 from GEO database,and analyzed the datasets through differentially expressed genes analysis,GO,and KEGG enrichment analysis.We found TRIM25 was significantly differentially expressed in both of datasets,and could be enriched in NF-?B signaling which suggested its potential role in regulation of this pathway.We confirmed the function of TRIM25 in the activation of TNF?-induced NF-?B signaling detected by luciferase assay,immunoblotting,and real-time PCR..Overexpression of TRIM25 promoted the degradation of I?B?,increased the transcription of NF-?B signaling downstream genes,and enhanced the activation of TNF?-induced NF-?B signaling.Conversely knockdown of TRIM25 had the opposite function.We found TRIM25 had interaction with TRAF2,and the interaction between TRIM25 and TRAF2 was enhanced after stimulated by TNFa from the results of immunoprecipitation Overexpression of TRIM25 enhanced the K63-linked polyubiquitination of TRAF2,and heightened the interaction between TRAF2 and TAK1 or KK? which further promoted the activation of NF-?B signaling.Knockdown of TRIM25 reduced the ubiquitination of TRAF2,and inhibited the interaction between TRAF2 and TAK1.These results indicated that TRIM25 could targeted TRAF2,and promoted the activation of TNF?-induced NF-?B signaling.In conclusion,this study focused on the E3 ubiquitin ligase CHIP and TRIM25,and rapidly screened the signalings influencing by CHIP and TRIM25 through bioinformatics analysis,and explored the mechanisms of CHIP up-regulated Wnt signaling by increasing the ubiquitination of LEF1,TRIM25 up-regulated TNF?-induced NF-?B signaling by increasing the ubiquitination of TRAF2 through multiple cytological experiments.They will be heplful to illustrate the pathogenic mechanism of SCAR16 caused by CHIP mutant,and the mechanism of TRIM25 in the regulation of innate immune response.