(188cp) Palmitate Directly Interacts with IRE1? to Induce Its Activation

The endoplasmic reticulum (ER) is the site for protein synthesis, folding and maturation. If these processes are disrupted, the Unfolded Protein Response (UPR) is activated to re-establish homeostasis. ER stress is sensed through IRE1, PERK and ATF6 sensor proteins. IRE1α is one of the ER stress sensor proteins that is activated during the UPR. IRE1α a transmembrane serine-threonine protein kinase/endoribonuclease known to be activated through dimerization of its luminal domain. Its failure to correct ER stress is implicated in the pathology of many diseases.

We have developed IRE1α^-/^- cell lines to investigate the effect of mutations in IRE1α and how they affect dimerization, activation and downstream signaling in the UPR. Molecular dynamic simulations predicted certain residues on the transmembrane (TM) domain and cytosolic domain (CD) that palmitate (PA) directly interacts with. Interaction by PA at these key residues plays an important role in the dimerization of IRE1α. Alanine scanning of TM-IRE1α aided by a bimolecular fluorescence complementation (BiFC) assay revealed important residues in IRE1α activation. The CD-IRE1α residues predicted to be involved in PA binding were mutated and expressed in insect Sf21 cells and the effects of these mutations were analyzed with a binding assay. This study provided evidence that PA activates IRE1α through a non-canonical luminal-domain independent mechanism.