The Endoplasmic Reticulum (ER) is a cellular compartment responsible for protein folding, lipid synthesis, and calcium storage. Physiological conditions such as elevated levels of free fatty acids (FFAs) and glucose, oxidative stress, and inflammatory cytokines are known to perturb ER homeostasis, leading to the accumulation of unfolded/misfolded proteins in the ER lumen. Especially, palmitic acid, a saturated FFA, is recognized to induce ER stress, in particular, with respect to two kinases proteins, PKR (cytosolic kinase) and IRE1α (Type-I transmembrane ER kinase), and to contribute to many diseases, e.g. cancer, diabetes, neurodegenerative and inflammatory diseases. However, the molecular mechanisms by which palmitate regulates the activities of the PKR and IRE1α proteins, the latter is a transmembrane (TM) protein, are not known. With the assistance of computational analysis, the biological and biochemical experiments showed that palmitic acid directly interacts with the kinase domains of both PKR and IRE1α. Palmitic acid bound to the αC-helix, an important structural feature for correct alignment of the catalytic residues, to regulate the enzymatic activity of these proteins. Furthermore, for a better understanding of the roles of palmitic acid on the IRE1α protein, the functional and structural roles of the TM domain were investigated. Mutational studies showed that Tryptophan (IRE1α-W457) serves as a driving force for the TM dimerization process and both the aromatic interaction and hydrogen bond formed by Trp457 are crucial for stabilizing the oligomerization of the TM domain, and possibly contributing to the palmitate-induced activity. Therefore, the current study could shed light into the molecular mechanisms by which palmitic acid mediates ER-stress induced diseases and further could lead to novel drug therapies.
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