PMID- 34995415
OWN - NLM
STAT- MEDLINE
DCOM- 20220608
LR  - 20221005
IS  - 1742-4658 (Electronic)
IS  - 1742-464X (Print)
IS  - 1742-464X (Linking)
VI  - 289
IP  - 11
DP  - 2022 Jun
TI  - Off-target inhibition of NGLY1 by the polycaspase inhibitor Z-VAD-fmk induces 
      cellular autophagy.
PG  - 3115-3131
LID - 10.1111/febs.16345 [doi]
AB  - 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, Ca(2+) 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. ENZYMES: 
      Peptide:N-glycanase1, Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase 
      [EC:3.5.1.52].
CI  - (c) 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf 
      of Federation of European Biochemical Societies.
FAU - Needs, Sarah H
AU  - Needs SH
AD  - School of Life, Health and Chemical Sciences, The Open University, Milton Keynes, 
      UK.
AD  - Reading School of Pharmacy, University of Reading, UK.
FAU - Bootman, Martin D
AU  - Bootman MD
AD  - School of Life, Health and Chemical Sciences, The Open University, Milton Keynes, 
      UK.
FAU - Grotzke, Jeff E
AU  - Grotzke JE
AD  - Yale University School of Medicine, New Haven, CT, USA.
FAU - Kramer, Holger B
AU  - Kramer HB
AD  - Department of Physiology, Anatomy and Genetics, University of Oxford, UK.
AD  - MRC London Institute of Medical Sciences, UK.
FAU - Allman, Sarah A
AU  - Allman SA
AUID- ORCID: 0000-0003-4904-4138
AD  - School of Life, Health and Chemical Sciences, The Open University, Milton Keynes, 
      UK.
AD  - Reading School of Pharmacy, University of Reading, UK.
AD  - Leicester School of Pharmacy, De Montfort University, Leicester, UK.
LA  - eng
GR  - WT_/Wellcome Trust/United Kingdom
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20220118
PL  - England
TA  - FEBS J
JT  - The FEBS journal
JID - 101229646
RN  - 0 (Peptides)
RN  - EC 3.4.22.- (Caspases)
RN  - EC 3.5.1.52 (Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase)
SB  - IM
MH  - Animals
MH  - Autophagy
MH  - Caspases
MH  - *Fibroblasts/metabolism
MH  - HEK293 Cells
MH  - Humans
MH  - Mice
MH  - Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine 
      Amidase/chemistry/genetics/metabolism
MH  - Peptides/metabolism
MH  - *Proteomics
PMC - PMC9304259
OTO - NOTNLM
OT  - NGLY1
OT  - Peptide: N-glycanase 1
OT  - Z-VAD-fmk
OT  - autophagosome proteomics
OT  - autophagy
COIS- The authors declare no conflict of interest.
EDAT- 2022/01/08 06:00
MHDA- 2022/06/09 06:00
PMCR- 2022/07/22
CRDT- 2022/01/07 17:27
PHST- 2021/09/14 00:00 [revised]
PHST- 2020/12/28 00:00 [received]
PHST- 2022/01/05 00:00 [accepted]
PHST- 2022/01/08 06:00 [pubmed]
PHST- 2022/06/09 06:00 [medline]
PHST- 2022/01/07 17:27 [entrez]
PHST- 2022/07/22 00:00 [pmc-release]
AID - FEBS16345 [pii]
AID - 10.1111/febs.16345 [doi]
PST - ppublish
SO  - FEBS J. 2022 Jun;289(11):3115-3131. doi: 10.1111/febs.16345. Epub 2022 Jan 18.