The inositol-requiring enzyme 1 (IRE1α) RNAse inhibitor, 4µ8C, is also a potent cellular antioxidant

Stanley M.H Chan,Ashton Bernard,Mark P Lowe,Terence P Herbert,Alyson A Miller

doi:10.1042/bcj20170678

Abstract

Inositol-requiring enzyme 1 alpha (IRE1α) is an endoplasmic reticulum (ER)-transmembrane endonuclease that is activated in response to ER stress as part of the unfolded protein response (UPR). Chronic activation of the UPR has been implicated in the pathogenesis of many common diseases including diabetes, cancer, and neurological pathologies such as Huntington's and Alzheimer's disease. 7-Hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde (4µ8C) is widely used as a specific inhibitor of IRE1α ribonuclease activity (IC50 of 6.89 µM in cultured cells). However, in this paper, we demonstrate that 4µ8C acts as a potent reactive oxygen species (ROS) scavenger, both in a cell-free assay and in cultured cells, at concentrations lower than that widely used to inhibit IRE1α activity. In vitro we show that, 4µ8C effectively decreases xanthine/xanthine oxidase catalysed superoxide production with an IC50 of 0.2 µM whereas in cultured endothelial and clonal pancreatic β-cells, 4µ8C inhibits angiotensin II-induced ROS production with IC50 values of 1.92 and 0.29 µM, respectively. In light of this discovery, conclusions reached using 4µ8C as an inhibitor of IRE1α should be carefully evaluated. However, this unexpected off-target effect of 4µ8C may prove therapeutically advantageous for the treatment of pathologies that are thought to be caused by, or exacerbated by, both oxidative and ER stress such as endothelial dysfunction and/or diabetes.

Highlights

The endoplasmic reticulum (ER) is the site for the synthesis and processing of secretory and membrane proteins
To formally demonstrate that angiotensin II (AngII)-generated superoxide production was through the activation of NADPH oxidases (NOX) rather than ER stress-induced inositol requiring enzyme 1α (IRE1α) activation, Mouse Insulinoma 6 (MIN6) and bEnd.3 cells were treated with AngII in the presence or absence of two NOX inhibitors, diphenyleneiodonium (DPI) and apocynin [30](Figure 2c and d)
Our data demonstrates that 4μ8C, at concentrations widely used to inhibit IRE1α activity, acts as a potent reactive oxygen species (ROS) scavenger both in a cell free assay and in cultured cells

Summary

Introduction

The endoplasmic reticulum (ER) is the site for the synthesis and processing of secretory and membrane proteins. IRE1α senses perturbations in ER homeostasis via its luminal domain which results in a conformational change [6,7] This in turn promotes oligomerisation and the activation of its cytoplasmic protein kinase and RNAse domain. 4μ8C is an aromatic aldehyde that binds to IRE1α’s RNAse domain and inhibits its activity This inhibitor has provided valuable insights into defining the mechanism of action of IRE1α in both cellular physiology and pathophysiology (e.g.[13,14,15,16,17,18,19]) . Superoxide counts were measured using a BMG Clariostar plate reader (BMG Labtech, Melbourne, Australia). After treatments superoxide counts were measured using a BMG Clariostar plate reader over 90 min (BMG Labtech, Melbourne, Australia; 45 cycles, 3 s per well). Differences were considered statistically significant at p < 0.05

Results

Discussion