"The endoplasmic reticulum (ER) is the site for protein folding and maturation. ER stressors, both physiological and pharmacological, can result in activation of the unfolded protein response (UPR). Palmitate, a saturated fatty acid, is one such ER stressor and leads to induction of the UPR. This is primarily through the activation of Inositol Requiring Enzyme-1 (IRE1) leading to splicing of XBP1 mRNA. However, the mechanism of this activation is unclear. With the aid of a bimolecular fluorescence complementation (BiFC) assay, we identified two crucial residues on the transmembrane domain (TM) of IRE1, S450 and W457, that are drivers of palmitate mediated activation. Previous research from our group suggested that IRE1 also has binding sites for palmitate on its cytosolic domain (CD). However, IRE1-CD protein expressed in E. coli was over-phosphorylated which possibly affected its binding to PA. To investigate this, we developed a protocol for expression and purification of wild type and mutant IRE1-CD protein in insect Sf21 cells. A fluorescence polarization based binding assay was performed to determine whether palmitate binds to residues on the IRE1-CD protein. Previously our laboratory demonstrated that palmitate induced the migration of cancer cells as well as transcription factors (TF) involved in epithelial-to-mesenchymal transition (EMT). Here, we investigated the role of IRE1 activation on these processes. Using CRISPR gene editing to generate IRE1 knockouts in liver and breast cancer cell lines, we observed that IRE1 mediates the upregulation in EMT-TFs, a decrease in the expression of the desmoplakin (DSP) protein, and an increase in the migration of liver and breast cancer cells. DSP is a critical component of desmosomes, which function to maintain the structural integrity at adjacent cell-cell contacts.In addition to migration, the effect of XBP1 splicing on metabolism has not been studied. We found the activation of IRE1-XBP1 is accompanied by changes in the metabolic genes involved in glycolysis, fatty acid oxidation, gluconeogenesis, and ceramide metabolism, suggesting that some of the metabolic effects of palmitate are mediated through IRE1. These results could have implications on the development of chemotherapeutic strategies. This study paves the way for further investigations into the far-reaching effects of activation of the UPR on cell survival, metabolism, and chemo-tolerance."--Pages ii-iii.
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