Screening Of Interacting Proteins For IRE1 By Yeast-Two-Hybrid And Its Role In The Apoptosis Of Hepatocytes

The Endoplasmic Reticulum(ER) resident protein Inosital Requiring Enzyme 1(IRE1) is the core sensor of ER stress, which senses the accumulation of unfolded protein in the ER lumen and transduces the signal to the rest part of the cell. Recent reports show that ER stress is associated with a lot of hepatic diseases, such as virus infection(HBV,HCV), diabetes-obese-related liver disease, alcoholic liver diseases,α1-antitrypsin deficiency and ischemia/reperfusion injury. The Unfolded Protein Response(UPR) induced by ER stress can reduce ER protein load through the induction of molecular chaperones and shut down of translation. However, persistent or intense ER stress result in apoptotic cell death. In order to elucidate the mechanism of ER stress induced apoptosis in hepatocytes, we screened the interacting proteins of IRE1 in a yeast-two-hybride system so as to give light to the investigation of ER stress in liver diseases.1. Screening of the interacting proteins of IRE1 in a yeast-two-hybrid system. Yeast-two-hybrid were employed to screen the interacting proteins for the N-termius(1-465aa) and C-terminus(468-977) of hIRE1αin a human testis cDNA library. The bait plasmids and the library cDNA were sequentially transfected into Saccharomyces cerevisiae AH109. Screening was carried out on a series of nutrition-selective plates, X-α-GAL assays and PCR assays. Yeast two-hybrid retransformation experiment was applied to verify the interactions. Results: Bait plasmids pGBKT7-IRE1N(1-465)and pGBKT7-IRE1C(468-977) were successfully constructed and the expression of fusion-proteins were correct. Tests for autonomous activation and toxicity were negative. The numbers of confirmed interactions were 8 for hIRE1α-N and 15 for hIRE1α-C. Retransformation experiment verified RACK1, transcript of the gene GNB2L1, a cytoplasmic located scaffold protein as a binding partner of the C terminus of IRE1.2. Verification of the interaction of IRE1 and RACK1 in mammalian cells and in vitro. (1) Construction of eukaryotic expression plasmids: 3XFLAG-IRE1C, GFPC3-RACK1. The construction was confirmed by restriction enzyme catalyzation and DNA sequencing. The plasmids were co-transfected into 293T cells and co-immunoprecipitation was applied to test the interaction of IRE1 and RACK1 in mammalian cells. (2) Prokaryote expression plasimds pGEX-6p2-RACK1,pGEX-6p1-IRE1C were constructed and transformed into E.coli BL21, the GST-fusion protein were purified by standard protocol. Fusion protein GST-RACK1 were catalyzed by PreCisionTM enzyme to produce the purified RACK1 protein. GST-Pull down assay were carried out to verify the in vitro binding of RACK1 to IRE1. (3) Eukaryotic expression plasmid GFPC3-IRE1WT were constructed for transfection HepG2 cells. The transfected HepG2 cells expressing fusion protein GFP-IRE1 WT were then used to do immunofluorescence experiment with anti-RACK1 mAb and Rodamine-conjugated anti-mouse secondary antibody. Results: In vivo and in vitro tests showed that RACK1 can bind to the C-terminus of IRE1. Immunofluorescence confirmed the co-locolization of IRE1 and RACK1, which give solid evidence for the interaction of the two proteins.3. RNAi tests for the function of the interaction between IRE1 and RACK1.(1)Transfect RACK1 SiRNA into HepG2 cells. Collect the cell lysates after 48h to test the interfering efficiency by western blot. The Si RNA treated and untreated cells were administered with thapsigargin to induce ER stress. Western blot were apllied to test the expression level of GRP78 and phosphorylated IRE1 to assess the activation of IRE1. RT-PCR assays were applied to test the expression and splice of XBP-1 mRNA. (2) TUNEL assays were applied to observe the effect on ER stress-mediated apoptosis by RACK1 SiRNA treatment. Results: The inhibition of RACK1 expression in HepG2 cells by SiRNA administration was successful. Our results confirmed that RACK1 depletion could suppress the activation of IRE1 under ER stress conditions, which inhibited ER stress mediated apoptosis of hepatocytes at the same time.Conclusion: Screening of interacting proteins of IRE1 by yeast-two-hybrid methods identified RACK1 as a novel binding partner. In vivo and in vitro assays confirmed the interaction. Immunofluorescence provided the direct evidence. Finally, RNAi experiments confirmed the function of RACK1's binding to IRE1 in that it could mediate the activation of IRE1 and downstream pathways such as the upregulation of GRP78 and the catalyzation of XBP-1 as well as induction of apoptosis under ER stress conditions, indicating RACK1 as a component of IRE1 signalling network and closely related to ER stress mediated apoptosis of hepatocytes. Further studies are needed for the role of ER stress in liver diseases.