(193ai) Novel Role of Transmembrane Domain of IRE1? Protein during Activation and its Implications in Progression of Cancer

The endoplasmic reticulum(ER) is the site for protein synthesis, folding and maturation. The unfolded protein response (UPR) is activated in response to a disruption of ER homeostasis. IRE1α is a transmembrane serine-threonine protein kinase/endoribonuclease and one of the ER stress sensor proteins that is activated during the UPR. Its failure to correct ER stress is implicated in the pathology of many diseases. The established method of activation of IRE1α is through dimerization mediated through its luminal domain.

Palmitate (PA) activates the UPR in various cell types. Long-chain fatty acids like palmitate (C16) are also known to induce epithelial-to-mesenchymal transition (EMT). An increase in free fatty acid levels is observed in various cancers (e.g. hepatocellular carcinoma) and the altered fatty acid metabolism is required to sustain tumor cells. However, the mechanism by which PA activates IRE1α to lead to EMT is unclear.

We hypothesize that unlike other ER stressors, PA activates IRE1α through a non-canonical luminal-domain independent mechanism. Specifically, we identified a W457 residue in the transmembrane domain of IRE1α that forms a part of the dimer interface on IRE1α. Mouse embryonic fibroblasts (MEF) IRE1α-/- knockout cells expressing wild-type (WT) IRE1α were more invasive than cells expressing IRE1α carrying a W457A mutation as demonstrated by wound healing studies. A bimolecular fluorescence (BiFC) assay was used to probe the importance of W457 in dimerization and activation of IRE1α. This assay uses the association of fragments of the Venus fluorescent protein driven by protein-protein interactions to demonstrate assembly of the IRE1α dimer. We confirmed that palmitate induces the dimerization of the transmembrane domain of IRE1α to activate IRE1α.

Studying the effect of fatty acids on the invasiveness and metastatic ability of cells through ER stress sensors like IRE1α provides insights into the biochemical changes induced by fatty acids that contribute to cancer progression.