Establishment And Application Of Ubiquitin Ligase Screening System Based On NanoBit Technolog

Objective: As the key protein of endoplasmic reticulum stress,XBP1 s play an important role in lipid metabolism.Considered the obscure stabilization of XBP1 s and the ubiquitination system participate in the unstable protein of it.Ubiquitination is an important post-translational modification that primarily regulates the stability and function of proteins.Ubiquitin ligase is the key enzyme to recognize substrate specificity in the process of ubiquitination.Currently,there is a lack of an efficient and simple method to quickly screen the specific ubiquitin ligase of the substrate.Therefore,this study aims to establish a ubiquitin ligase screening system,find the ubiquitin ligase of XBP1 s,and further explore the effect of XBP1 s protein homeostasis on lipid metabolism.Methods: In this study,NanoBit technology was used to establish a ubiquitin ligase screening system,through which XBP1 s ubiquitin ligase was screened and verified.Then,the effect of ubiquitin ligase of XBP1 s on lipid metabolism under endoplasmic reticulum stress was explored in hepatocytes and finally verified in C.elegans.Results: We developed a ubiquitin ligase screening system based on the HiB-Ub cell line and successfully identified the ubiquitin ligases TRAF2 and PJA1 for XBP1 s.Mechanistically TRAF2 and PJA1 can interact with the XBP1 s directly.In addition,I originally discover that PJA1 can ubiquitinate the residue lysine 72 of XBP1 s.Our results show that overexpression of TRAF2 and PJA1 can decrease the expression of XBP1 s and SCD,a protein downstream of XBP1 s,under palmitic acid treatment.In the hepatocyte,lipid droplets can be reduced by TRAF2 and PJA1,respectively.Furthermore,the knockdown of the analog protein of TRAF2 and PJA1 in worms also increases the xbp1 signaling and lipids accumulation.Conclusion: We uncover a conserved signaling cascade that TRAF2 and PJA1 ubiquitinate the XBP1 s and blunt its’ function.This discovery may suggest therapeutic strategies for disorders associated with XBP1 s on the one hand and complements the endoplasmic reticulum stress pathway on the other.