Off-target inhibition of NGLY1 by the poly-caspase inhibitor Z-VAD-fmk induces cellular autophagy

Needs, S. H. ORCID: https://orcid.org/0000-0003-3407-9637, Bootman, M. D., Grotzke, J. E., Kramer, H. B. and Allman, S. A. (2022) Off-target inhibition of NGLY1 by the poly-caspase inhibitor Z-VAD-fmk induces cellular autophagy. The FEBS Journal, 289 (11). pp. 3115-3131. ISSN 1742-464X doi: 10.1111/febs.16345

Abstract/Summary

The pan-caspase 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. Activation of ER stress markers or induction of reactive oxygen species (ROS) are 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 proteomics analysis reveal 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.

Item Type	Article
URI	https://reading-clone.eprints-hosting.org/id/eprint/102320
Identification Number/DOI	10.1111/febs.16345
Refereed	Yes
Divisions	Life Sciences > School of Chemistry, Food and Pharmacy > School of Pharmacy > Division of Pharmacology
Publisher	Wiley
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