![]() Despite binding with similar affinity to IRE1, most kinase inhibitors allosterically activate the RNase, while some are neutral, and a minority allosterically inhibits the RNase 19, 20. Uniquely, IRE1 kinase inhibitors are capable of allosterically modulating IRE1’s RNase activity in opposite directions. Targeting either the kinase or RNase domain with small molecules has been shown to inhibit IRE1 RNase activity 9, 19, 20. IRE1 has recently gained interest as a potential therapeutic target 17, 18. RIDD is thought to reduce the protein-folding burden on the ER 13, as well as to modulate specific cellular functions, including apoptosis 14, 15 and lysosomal function 16. Additionally, the IRE1 RNase drives the degradation of a subset of ER-localized mRNAs and microRNAs through an as yet incompletely understood process called Regulated IRE1-Dependent Decay (RIDD) 13. The transcript is then translated into a potent transcription factor, which activates numerous genes that help relieve ER stress 12. The IRE1 RNase specifically cleaves the double hairpin motif of unspliced XBP1 mRNA, leading, after ligation of the exons by the RtcB tRNA ligase 11, to a spliced XBP1 (XBP1s) mRNA. The B2B dimeric and oligomeric IRE1 complexes are stabilized by phosphorylation of the kinase activation loop, which in human IRE1 occurs on residues S724, S726, S729, making phosphorylated IRE1 (IRE1-3P) the most active form of the RNase enzyme 8, 9, 10. These clusters, visualized by fluorescence tagging as foci within cells, are proposed to be the physiologically relevant forms of active IRE1 7.Įvidence so far indicates that the kinase activity of IRE1 is highly directed toward trans-autophosphorylation. Furthermore, IRE1 forms distinct supermolecular clusters under certain conditions. In the B2B dimer, the kinase domains dimerize with their ATP-binding pockets facing away from each other and provide the building blocks for higher-order IRE1 oligomers 5, 7. The cytoplasmic domain has been shown to self-associate via different surface contacts, where a back-to-back (B2B) configuration is believed to fulfill the essential structural requirements for RNase activation 5, 6. The association of the lumenal domain promotes dimerization and oligomerization of the cytoplasmic kinase-RNase tandem domain. ![]() The lumenal domain senses unfolded proteins in the ER through indirect and direct mechanisms: as a result, it undergoes dimerization and subsequent oligomerization 3, 4. IRE1 integrates multiple functional domains into a single ER-membrane-spanning protein. Of these, IRE1 is the most evolutionarily conserved 2. ![]() In metazoans, the UPR comprises interplaying signaling branches transmitted through three ER transmembrane proteins: Inositol-Requiring Enzyme 1 (IRE1), Protein kinase R-like Endoplasmic Reticulum Kinase (PERK), and Activating Transcription Factor 6 (ATF6). The Unfolded Protein Response (UPR) is a highly regulated intracellular network that alleviates endoplasmic reticulum (ER) stress caused by elevated levels of misfolded proteins in the ER lumen 1. Our findings reveal exquisitely precise interdomain regulation within IRE1, advancing the mechanistic understanding of this important enzyme and its investigation as a potential small-molecule therapeutic target. This class of activators binds IRE1 in the kinase front pocket, leading to a distinct conformation of the activation loop. In this study, we describe a class of ATP-competitive RNase activators possessing high selectivity and strong cellular activity. Previous allosteric RNase activators display poor selectivity and/or weak cellular activity. Known ATP-competitive small-molecule IRE1 kinase inhibitors either allosterically disrupt or stabilize the active dimeric unit, accordingly inhibiting or stimulating RNase activity. Excess unfolded proteins in the ER lumen induce dimerization and oligomerization of IRE1, triggering kinase trans-autophosphorylation and RNase activation. IRE1 spans the endoplasmic reticulum membrane, comprising a sensory lumenal domain, and tandem kinase and endoribonuclease (RNase) cytoplasmic domains. ![]() Inositol-Requiring Enzyme 1 (IRE1) is an essential component of the Unfolded Protein Response.
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