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Autophagosome Proteomics of HEK293 cells

ABSTRACT: 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 during ER-associated degradation (ERAD). Pharmacological N-glycanase inhibition by Z-VAD-fmk or siRNA knockdown (KD) induces GFP-LC3 positive puncta in HEK293 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 similar autophagosomal protein content. 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.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hek-293 Cell

SUBMITTER: Holger Kramer

LAB HEAD: Holger Kramer

PROVIDER: PXD020367 | Pride | 2022-01-12

REPOSITORIES: Pride

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Project description:Isolated human hepatocytes are used in all facets of liver research, from the clinical management of liver failure to in vitro studies of drug disposition. Cryopreservation provides a reliable source of hepatocytes, but the associated cellular stress causes a highly variable and heterogeneous loss of a differentiated phenotype, manifested by a decreased ability to form cell-matrix and cell-cell interactions. We reasoned that this problem could be mitigated at the post-thawing stage, which would increase the availability of well-functioning human hepatocytes. We applied quantitative global proteomics to analyze the differences between attached and non-attached fractions of cryopreserved human hepatocyte batches. Hepatocytes that were unable to attach to a collagen matrix showed many signs of cellular stress, including a glycolytic phenotype and activation of the heat shock response, with increased apoptosis activation as the ultimate consequence. Further analysis of the activated stress pathways revealed an increase in early apoptosis immediately after thawing, hinting at the possibility of stress reversal. Therefore, we transiently treated the cells with compounds aimed at decreasing cellular stress via different mechanisms. We found that brief exposure to the pan-caspase apoptosis inhibitor Z-VAD-FMK restored the ability to attach to collagen, and promoted a differentiated morphology with increased metabolic function. Further, Z-VAD-FMK treatment did not alter hepatocyte protein expression, suggesting that these ‘rescued’ cells would be suitable for applications where hepatocytes of high quality are required. Conclusion: Cryopreserved human hepatocytes are affected by considerable amounts of cellular stress, often resulting in loss of differentiation with a decreased ability to form cell-matrix and cell-cell interactions. This can be alleviated by brief apoptosis inhibition post-thawing, substantially improving key morphological and functional properties of these cells.

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	20171003_HK_VC1_TR01.raw	Raw
	20171003_HK_VC2_TR01.raw	Raw

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Autophagosome Proteomics of HEK293 cells

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