Structure, Mechanism and Chemical Modulation of the Protein Kinase-nuclease Dual-enzyme IRE1
type: CreativeWork
description: Perturbations that derail the proper folding and assembly of proteins in the endoplasmic retriculum (ER) cause misfolded protein accrual in the ER – a toxic condition known as ER stress. The Unfolded Protein Response (UPR) is a signaling system evolved to detect and rectify ER stress. The work I present herein pertains to the most ancient member of the ER stress transducers, IRE1. ER stress stimulates IRE1 to activate a UPR-dedicated transcription factor called XBP1 in metazoans (or HAC1 in yeast) to bolster the productive capacity of the ER and purge misfolded proteins from the ER. To activate XBP1/HAC1, IRE1 cleaves XBP1/HAC1 mRNA twice to eliminate an inhibitory intron using a dormant nuclease function in its cytoplasmic effector region (IRE1cyto). My focus was to understand the mechanism of XBP1/HAC1 activation by IRE1, the regulation of IRE1 function and the manipulation of IRE1 signaling output using chemical tools. To better understand IRE1 mechanism, I determined the crystal structure of IRE1cyto bound to ADP. Structural and mutational analyses uncovered a probable novel IRE1 nuclease active site, allowing a catalytic mechanism of RNA cleavage to be inferred. Further genetic and biophysical experiments revealed that the ordered sequence of events: autophosphorylation, nucleotide binding and dimerization; orchestrates the assembly of the IRE1 nuclease active site to potentiate nuclease function. The flavanol quercetin was identified in a chemical screen as a potent stimulator of IRE1 nuclease output. To understand the mechanism of action of quercetin, I determined the crystal structure of IRE1cyto in complex with quercetin and ADP. Quercetin docked to a novel ligand binding site, termed the Q-site, at the interface of IRE1 dimers. Biophysical and genetic analyses revealed that quercetin engagement of the Q-site promotes IRE1 dimerization, thereby enhancing IRE1 nuclease activity. To gain insight on how IRE1 recognizes RNA, I performed bioinformatic analysis t…
abstract: Perturbations that derail the proper folding and assembly of proteins in the endoplasmic retriculum (ER) cause misfolded protein accrual in the ER – a toxic condition known as ER stress. The Unfolded Protein Response (UPR) is a signaling system evolved to detect and rectify ER stress. The work I present herein pertains to the most ancient member of the ER stress transducers, IRE1. ER stress stimulates IRE1 to activate a UPR-dedicated transcription factor called XBP1 in metazoans (or HAC1 in yeast) to bolster the productive capacity of the ER and purge misfolded proteins from the ER. To activate XBP1/HAC1, IRE1 cleaves XBP1/HAC1 mRNA twice to eliminate an inhibitory intron using a dormant nuclease function in its cytoplasmic effector region (IRE1cyto). My focus was to understand the mechanism of XBP1/HAC1 activation by IRE1, the regulation of IRE1 function and the manipulation of IRE1 signaling output using chemical tools. To better understand IRE1 mechanism, I determined the crystal structure of IRE1cyto bound to ADP. Structural and mutational analyses uncovered a probable novel IRE1 nuclease active site, allowing a catalytic mechanism of RNA cleavage to be inferred. Further genetic and biophysical experiments revealed that the ordered sequence of events: autophosphorylation, nucleotide binding and dimerization; orchestrates the assembly of the IRE1 nuclease active site to potentiate nuclease function. The flavanol quercetin was identified in a chemical screen as a potent stimulator of IRE1 nuclease output. To understand the mechanism of action of quercetin, I determined the crystal structure of IRE1cyto in complex with quercetin and ADP. Quercetin docked to a novel ligand binding site, termed the Q-site, at the interface of IRE1 dimers. Biophysical and genetic analyses revealed that quercetin engagement of the Q-site promotes IRE1 dimerization, thereby enhancing IRE1 nuclease activity. To gain insight on how IRE1 recognizes RNA, I performed bioinformatic analysis t…
author: Lee, Kenneth
language: en_ca
keywords: Unfolded Protein Response, ER stress
date published: 2012-12-05
date modified: 2026-07-27T21:28:22Z
provider: University of Toronto
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Toronto

PropertyValue
property ID: Handle
value: http://hdl.handle.net/1807/33815