Regulation of the unfolded protein response via S-nitrosylation of sensors of endoplasmic reticulum stress.

Ryosuke Nakato,Yuki Kaneko,Akari Konishi,Takashi Uehara,Mari Shibata,Takao Iwawaki,Tomohiro Nakamura,Stuart A Lipton,Yu Ohkubo

doi:10.1038/srep14812

Abstract

Protein S-nitrosylation modulates important cellular processes, including neurotransmission, vasodilation, proliferation, and apoptosis in various cell types. We have previously reported that protein disulfide isomerase (PDI) is S-nitrosylated in brains of patients with sporadic neurodegenerative diseases. This modification inhibits PDI enzymatic activity and consequently leads to the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) lumen. Here, we describe S-nitrosylation of additional ER pathways that affect the unfolded protein response (UPR) in cell-based models of Parkinson’s disease (PD). We demonstrate that nitric oxide (NO) can S-nitrosylate the ER stress sensors IRE1α and PERK. While S-nitrosylation of IRE1α inhibited its ribonuclease activity, S-nitrosylation of PERK activated its kinase activity and downstream phosphorylation/inactivation or eIF2α. Site-directed mutagenesis of IRE1α(Cys931) prevented S-nitrosylation and inhibition of its ribonuclease activity, indicating that Cys931 is the predominant site of S-nitrosylation. Importantly, cells overexpressing mutant IRE1α(C931S) were resistant to NO-induced damage. Our findings show that nitrosative stress leads to dysfunctional ER stress signaling, thus contributing to neuronal cell death.

Highlights

The endoplasmic reticulum (ER) is involved in many essential cellular processes, including calcium homeostasis, steroid synthesis, protein folding and maturation, and quality control of newly synthesized proteins[1,2]
Because HRD1 mRNA expression is known to be dependent on XBP1, it is possible that NO regulates this branch of the unfolded protein response (UPR) via IRE1α 21
Previous reports had shown that NO reacts with protein disulphide isomerase (PDI) to form SNO–PDI in the ER lumen of human brains obtained from patients with several neurodegenerative diseases characterized by abnormal protein accumulation, including Parkinson’s disease (PD)[20]

Summary

Introduction

The endoplasmic reticulum (ER) is involved in many essential cellular processes, including calcium homeostasis, steroid synthesis, protein folding and maturation, and quality control of newly synthesized proteins[1,2]. Severe cellular stress, engendered by hypoxia, energy deprivation, or exposure to excessive reactive oxygen or nitrogen species (including nitric oxide (NO)), can lead to ER stress Such stress triggers the unfolded protein response (UPR)[5,6,7,8], which, if terminated within a moderate amount of time, can lead to cytoprotection. The three major ER stress-sensing proteins are PKR-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6)[13,14,15] These sensors transmit signals from the ER to the cytoplasm or nucleus, and activate the following three pathways: (i) suppression of protein translation to halt the production of more unfolded proteins, (ii) induction of genes encoding ER molecular chaperones to facilitate protein folding, and (iii) activation of ER-associated degradation (ERAD) to decrease the accumulation of unfolded proteins in the ER16. We discovered that a number of ER stress-sensing proteins are S-nitrosylated, suggesting a novel regulatory mechanism for UPR activation

Methods

Results

Conclusion