NGLY1 knockdown or pharmacological inhibition induces cellular autophagy
| Autor: | Jeff E. Grotzke, Sarah Needs, Martin D. Bootman, Sarah Allman, Holger Kramer |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
chemistry.chemical_classification
0303 health sciences Reactive oxygen species Gene knockdown Endoplasmic reticulum Autophagy HEK 293 cells Endoplasmic-reticulum-associated protein degradation Cell biology 03 medical and health sciences 0302 clinical medicine chemistry Downregulation and upregulation parasitic diseases Unfolded protein response biological phenomena cell phenomena and immunity 030217 neurology & neurosurgery 030304 developmental biology |
| Zdroj: | bioRxiv |
| DOI: | 10.1101/2020.12.05.400481 |
| Popis: | SummaryPan-caspase inhibitor Z-VAD-fmk acts as an inhibitor of peptide:N-glycanase (NGLY1); an endoglycosidase which cleavesN-linked glycans from glycoproteins exported from the endoplasmic reticulum during ER-associated degradation (ERAD). PharmacologicalN-glycanase inhibition by Z-VAD-fmk or siRNA knockdown (KD) induces GFP-LC3 positive puncta in HEK 293 cells. Activation of ER stress markers or reactive oxygen species (ROS) induction are not observed. In NGLY1 inhibition or KD, upregulation of autophagosome formation without impairment of autophagic flux are observed. Enrichment and proteomics analysis of autophagosomes after Z-VAD-fmk treatment or NGLY1 KD reveals comparable autophagosomal protein content. 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 and Q-VD-OPh represents an alternative caspase inhibitor free from this limitation. |
| Databáze: | OpenAIRE |
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