(72d) The Roles of Palmitate On IRE1alpha Enzymatic Activities

IRE1α (inositol-requiring enzyme 1α) is the most highly conserved signaling node of the unfolded protein response (UPR). IRE1α consists of an N-terminal luminal domain and C-terminal cytosolic domain containing kinase/endoribonuclease activity, and are connected by a signal helix transmembrane domain. Upon ER stress, the signal in the luminal domain is transduced to the cytosol by sequential dimerization/multimerization, trans-autophosphorylation and activation of its endoribonuclease. The activated IRE1α splices Xbp1(X-box binding protein 1) mRNA generating an active Xbp1s transcriptional activator that upregulates expression of UPR target genes. It is well known that palmitate, C16-saturated fatty acid, induces ER stress by stimulating IRE1α activities in the cells. However, the molecular mechanism by which palmitate activates IRE1α is not well understood. Our group previously showed that palmitate directly interacts with the cytosolic domain of the IRE1α. We address whether palmitate regulates the enzymatic activities of IRE1α-CD protein using in-vitro kinase and RNase assays. Computational molecular dynamics simulations suggested several potential palmitate-binding sites (R611, R635, R687, K716, R722, R727, and R728) which are mainly located in the activation loop, dimerization interfaces, and αC-helix. We tested whether mutations of these sites are able to alter the palmitate-binding as well as the enzymatic activities of IRE1α-CD protein. The mutation analyses suggested that palmitate plays a role as an allosteric activator of IRE1α.  We further introduced the mutant genes into the IRE1^-/--MEF (Mouse Embryonic Fibroblast) cells, confirming that mutation blocks the palmitate-mediated activation of IRE1α. Therefore, these findings suggest that palmitate is an endogenous cytosplamic ligand that may activate the signals from the ER lumen to modulate IRE1α activities. In addition, the results provide insights into the development of drugs that modify IRE1α activities, and the signaling of the UPR.