Off‐target inhibition of NGLY1 by the polycaspase inhibitor Z‐VAD‐fmk induces cellular autophagy

Sarah H Needs,Martin D Bootman,Holger B Kramer,Jeff E Grotzke,Sarah A Allman

doi:10.1111/febs.16345

Abstract

The polycaspase inhibitor Z‐VAD‐fmk acts as an inhibitor of peptide: N‐glycanase (NGLY1), an endoglycosidase which cleaves N‐linked glycans from glycoproteins exported from the endoplasmic reticulum (ER) during ER‐associated degradation (ERAD). Both pharmacological N‐glycanase inhibition by Z‐VAD‐fmk and siRNA‐mediated knockdown (KD) of NGLY1 induce GFP‐LC3‐positive puncta in HEK 293 cells. The activation of ER stress markers or induction of reactive oxygen species (ROS) is not observed under either condition. Moreover, Ca2+ handling is unaffected when observing release from intracellular stores. Under conditions of pharmacological NGLY1 inhibition or NGLY1 KD, upregulation of autophagosome formation without impairment of autophagic flux is observed. Enrichment of autophagosomes by immunoprecipitation (IP) and mass spectrometry‐based proteomic analysis reveals comparable autophagosomal protein content. Gene ontology analysis of proteins enriched in autophagosome IPs shows overrepresentation of factors involved in protein translation, localization and targeting, RNA degradation and protein complex disassembly. Upregulation of autophagy represents a cellular adaptation to NGLY1 inhibition or KD, and ATG13‐deficient mouse embryonic fibroblasts (MEFs) show reduced viability under these conditions. In contrast, treatment with pan‐caspase inhibitor, Q‐VD‐OPh, does not induce cellular autophagy. Therefore, experiments with Z‐VAD‐fmk are complicated by the effects of NGLY1 inhibition, including induction of autophagy, and Q‐VD‐OPh represents an alternative caspase inhibitor free from this limitation.EnzymesPeptide:N‐glycanase1, Peptide‐N(4)‐(N‐acetyl‐beta‐glucosaminyl)asparagine amidase [EC:3.5.1.52].

Highlights

The peptide fluoromethyl ketone inhibitor Z-VAD-fmk has been applied extensively to the study of caspases and the central role of this important group of cysteine proteases in apoptosis
We were interested in investigating the cellular effects of inhibition of NGLY1 by Z-VAD-fmk, a known offtarget for this pan-caspase inhibitor. siRNA-mediated gene knock-down (KD) in HEK 293 cells leads to a significant reduction in NGLY1 transcript levels (Fig. 1a) while cell viability remains unaffected (Fig. 1b)
Intracellular Ca2+ handling is unaffected by treatment with Z-VAD-fmk, Q-VD-OPh or NGLY1 KD In order to assess whether intracellular Ca2+ signaling is affected by Z-VAD-fmk or Q-VD-OPh inhibitor treatment, we studied whether release of Ca2+ from intracellular stores is affected under these conditions

Summary

Introduction

The peptide fluoromethyl ketone inhibitor Z-VAD-fmk has been applied extensively to the study of caspases and the central role of this important group of cysteine proteases in apoptosis. Utilization as a tool in cellular research continues to date, despite the recognition that Z-VAD-fmk interacts with several off-targets These off-targets include other cysteine proteases, such as the cathepsins [2] and calpains [3], and the amidase peptide:N-glycanase 1 (NGLY1) [4]. The investigators went on to show that autocrine production of TNF was involved in this process and was mediated by the PKC-MAPKs-AP1 pathway They demonstrated that the NF-kB pathway exerts a protective function against necroptosis. Despite such disparate cellular outcomes for different caspase inhibitors, the precise mechanistic reasons for varying outcomes with different inhibitors are often unclear. It should be noted that significant differences in selectivity between fmk-based and alternative inhibitors itself may explain differences in engagement of cellular off-targets

Objectives

Methods

Results

Conclusion