Abstract

Inositol-Requiring Enzyme 1 (IRE1) is an essential component of the Unfolded Protein Response. IRE1 spans the endoplasmic reticulum membrane, comprising a sensory lumenal domain, and tandem kinase and endoribonuclease (RNase) cytoplasmic domains. Excess unfolded proteins in the ER lumen induce dimerization and oligomerization of IRE1, triggering kinase trans-autophosphorylation and RNase activation. Known ATP-competitive small-molecule IRE1 kinase inhibitors either allosterically disrupt or stabilize the active dimeric unit, accordingly inhibiting or stimulating RNase activity. Previous allosteric RNase activators display poor selectivity and/or weak cellular activity. In this study, we describe a class of ATP-competitive RNase activators possessing high selectivity and strong cellular activity. This class of activators binds IRE1 in the kinase front pocket, leading to a distinct conformation of the activation loop. 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.

Highlights

  • Inositol-Requiring Enzyme 1 (IRE1) is an essential component of the Unfolded Protein Response

  • After a 4-h incubation, G-9807 and G-1749 significantly increased the cellular levels of XBP1s mRNA (Fig. 1c) and decreased those of DGAT2 (Supplementary Fig. 1c), indicating compound-induced stimulation of IRE1 RNase activity

  • RNase activator G-1749 did not increase the dimeric population (Fig. 1e). These results suggest that while G-9807 acts as an allosteric RNase activator by stabilizing dimerization, G-1749 operates through a distinct mode, without altering the monomer-dimer equilibrium of IRE1

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Summary

Introduction

Inositol-Requiring Enzyme 1 (IRE1) is an essential component of the Unfolded Protein Response. IRE1 spans the endoplasmic reticulum membrane, comprising a sensory lumenal domain, and tandem kinase and endoribonuclease (RNase) cytoplasmic domains. Excess unfolded proteins in the ER lumen induce dimerization and oligomerization of IRE1, triggering kinase trans-autophosphorylation and RNase activation. We describe a class of ATP-competitive RNase activators possessing high selectivity and strong cellular activity. This class of activators binds IRE1 in the kinase front pocket, leading to a distinct conformation of the activation loop. 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]. 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]

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