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CALCOCO1 acts with VAMP-Associated Proteins to mediate ER-phagy

ABSTRACT: To explore the interaction partners of CALCOCO1, we immunoprecipitated EGFP-CALCOCO1 from HEK 293 cells’ lysate and identified the bound proteins by mass spectrometry. The identified proteins were then filtered against GFP control.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Jack-Ansgar Bruun

LAB HEAD: Terje Johansen

PROVIDER: PXD018894 | Pride | 2020-06-02

REPOSITORIES: Pride

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Project description:Liver sinusoidal endothelial cells (LSECs) are specialized fenestrated scavenger endothelial cells involved in the elimination of modified plasma proteins and tissue turnover waste macromolecules from blood. LSECs also participate in liver immune responses. A challenge when studying LSEC biology is the rapid loss of the in vivo cell phenotype in culture. In this study, we have examined biological processes and pathways affected during early-stage primary culture of rat LSECs and checked for cell responses to the pro-inflammatory cytokine interleukin (IL)-1 and the anti-inflammatory drug dexamethasone. Methods: LSECs from male Sprague Dawley rats were cultured on type I collagen in a 5% oxygen atmosphere in DMEM with serum-free supplements for 2 and 24 h. Quantitative proteomics using tandem mass tag technology were used to examine proteins in cells and supernatants. Validation was done with ELISA and multiplex immunobead assays, cell ultrastructure was examined by scanning electron microscopy, and scavenger function by quantitative endocytosis assays. Results: LSECs cultured for 24 h showed a characteristic pro-inflammatory phenotype both in the presence and absence of IL-1, displaying upregulation of cellular responses to cytokines and interferon-, cell-cell adhesion, and glycolysis, and downregulation of membrane/endocytosis receptors (MCAM, STAB1, STAB2, LYVE1, CLEC4G) and proteins involved in pyruvate metabolism, citric acid cycle, fatty acid elongation, amino acid metabolism, and oxidation-reduction processes. Dexamethasone improved LSEC viability in culture and repressed the culture-induced stimulation of glycolysis, inflammatory and immune regulatory pathways, and secretion of pro-inflammatory cytokines while increasing IL-10 secretion compared to time-matched control. The number of sieve plates in LSECs was reduced at 24 h both in the presence and absence of dexamethasone but the dexamethasone-treated cells showed a more quiescent phenotype. Conclusion: Rat LSECs become activated towards a pro-inflammatory phenotype during early culture. Dexamethasone represses LSEC activation and improves cell viability.

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CALCOCO1 acts with VAMP-Associated Proteins to mediate ER-phagy

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