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

Needs, Sarah H.; Bootman, Martin; Grotzke, Jeff E.; Kramer, Holger B. and Allman, Sarah A. (2022). Off‐target inhibition of NGLY1 by the poly‐caspase inhibitor Z‐VAD‐fmk induces cellular autophagy. FEBS Journal (Early Access).

DOI: https://doi.org/10.1111/febs.16345

Abstract

The pan-caspase inhibitor Z-VAD-fmk acts as an inhibitor of peptide:N-glycanase (NGLY1); an endoglycosidase which cleaves N-inked 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.

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