Signaling Mechanism of Mammalian Endoplasmic Reticulum Stress Sensor IRE1

The endoplasmic reticulum (ER) responds to the accumulation of misfolded proteins in its lumen (termed ER stress) by activating collective signal transduction pathways, called unfolded protein response (UPR). The UPR alleviates ER stress through its translational and transcriptional outputs carried out by three distinct ER stress sensors: PERK, ATF6 and IRE1. IRE1 is the best characterized UPR signal transduction molecule, and it is conserved from yeast to mammals. UPR signaling promotes apoptotic cell death if ER stress is not alleviated. However, the exact mechanism of how ER stress induces apoptosis remains unclear. Particularly, each branch of the UPR exerts opposing effects on cell survival under ER stress. To distinguish the effect of each branch of the UPR, we took a chemical-genetic approach, where we artificially prolonged IRE1 signaling by a small molecule, and observed enhanced cell survival under ER stress. Our data suggests a causal link between the duration of UPR branch signaling and life or death cellular decision. To further dissect IRE1 signaling upon prolonged ER stress, we developed a visual assay to assess IRE1 activation in cells. IRE1 is activated by misfolded protein direct binding to its lumenal domain, which then transduces the signal to its cytosolic kinase and endoribonuclease domains. Using the in vivo imaging approach and an in vitro biochemical assay, we demonstrated that IRE1 oligomerizes at the ER membrane upon misfolded protein accumulation, which in turn activates it kinase and RNase activities. IRE1 signaling attenuation correlates perfectly with its oligomers dissociation, suggesting a built-in timer within IRE1 that may be important in switching the UPR from the initially cytoprotective phase to the apoptotic mode. We next took an unbiased proteomic approach to address IRE1 signal attenuation mechanism using mass spectrometry analysis. We identified over 400 proteins and compiled a list of interesting and potential IRE1 interacting partners and modulators