Palmitate Effect on the Trasmembrane Domain of IRE1α Protein

Abstract

The Inositol-requiring kinase 1α protein (IRE1α) is a transmembrane protein kinase essential for the endoplasmic reticulum (ER) unfolded protein response that promotes cell survival by reducing the levels of misfolded protein. IRE1α consists of an N-terminal luminal domain, a single-pass transmembrane spanning segment and a cytosolic region which contains a kinase domain and an endoribonuclease (RNase) domain. Under conditions of ER stress, the transmembrane protein IRE1α oligomerizes to activate its cytoplasmic kinase and RNase domains. Recently, we showed that palmitate is involved in ER stress by increasing the kinase and RNase activities of IRE1α in human hepatocellular carcinoma (HepG2) cells. From our previous studies of palmitate in the membrane, palmitate aligns with the lipids. It is expected that palmitate will assume a similar conformation with the transmembrane (TM) domain, however, it is unclear whether palmitate binds directly to the TM domain or indirectly alter the structure of the bilayer surrounding the TM domain, thereby leading to enhanced association between the TM domains.We combined experiments with molecular dynamic (MD) simulations to investigate the properties of the TM domain, the role of palmitate on IRE1α dimerization and to elucidate potential interactions between palmitate and TM domain. Since structural information of the TM domain of IRE1α is not available, we experimentally determined whether the predicted TM domain forms α-helical secondary structure in DOPC liposome using circular dichroism. In support of the MD simulations, we assessed whether palmitate enhances TM dimerization in DOPC micelles using Forster resonance-energy transfer. Further mutation study of the TM domain provided information of how palmitate is involved in modulating the cellular activity of IRE1α mediated by the TM domain. Finally, the computational and experimental studies provided new insight into the role of palmitate on IRE1α activity during ER stress.