YIPF3 and YIPF4 regulate autophagic turnover of the Golgi apparatus
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ABSTRACT:
SUBMITTER: Mr. Shinri Kitta
PROVIDER: S-SCDT-10_1038-S44318-024-00131-3 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:The Golgi apparatus is an essential organelle of the secretory pathway in eukaryotic cells. It processes secretory and transmembrane proteins and orchestrates their transport to other endomembrane compartments or the plasma membrane. The Golgi apparatus thereby shapes the cell surface, controlling cell polarity, cell-cell communication, and immune signaling. The cytosolic face of the Golgi hosts and regulates signaling cascades, impacting most notably the DNA damage response and mitosis. These essential functions strongly depend on Golgi protein homeostasis and Golgi integrity. Golgi fragmentation and consequent malfunction is associated with neurodegenerative diseases and certain cancer types. Recent studies provide first insight into the critical role of ubiquitin signaling in maintaining Golgi integrity and in Golgi protein quality control. Similar to well described pathways at the endoplasmic reticulum, ubiquitin-dependent degradation of non-native proteins prevents the accumulation of toxic protein aggregates at the Golgi. Moreover, ubiquitination regulates Golgi structural rearrangements in response to cellular stress. Advances in elucidating ubiquitination and degradation events at the Golgi are starting to paint a picture of the molecular machinery underlying Golgi (protein) homeostasis.
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Project description:Ceramidases hydrolyze ceramides into sphingosine and fatty acids, with sphingosine being further metabolized into sphingosine-1-phosphate (S1P); thus, ceramidases control the levels of these bioactive sphingolipids in cells and tissues. Neutral ceramidase (nCDase) is highly expressed in colorectal tissues, and a recent report showed that nCDase activity is involved in Wnt/β-catenin signaling. In addition, the inhibition of nCDase decreases the development and progression of colorectal tumor growth. Here, to determine the action of nCDase in colorectal cancer cells, we focused on the subcellular localization and metabolic functions of this enzyme in HCT116 cells. nCDase was found to be located in both the plasma membrane and in the Golgi apparatus, but it had minimal effects on basal levels of ceramide, sphingosine, or S1P. Cells overexpressing nCDase were protected from the cell death and Golgi fragmentation induced by C6-ceramide, and they showed reduced levels of C6-ceramide and higher levels of S1P and sphingosine. Furthermore, compartment-specific metabolic functions of the enzyme were probed using C6-ceramide and Golgi-targeted bacterial SMase (bSMase) and bacterial ceramidase (bCDase). The results showed that Golgi-specific bCDase also demonstrated resistance against the cell death stimulated by C6-ceramide, and it catalyzed the metabolism of ceramides and produced sphingosine in the Golgi. Targeting bSMase to the Golgi resulted in increased levels of ceramide that were attenuated by the expression of nCDase, also supporting its ability to metabolize Golgi-generated ceramide. These results are critical in understanding the functions of nCDase actions in colorectal cancer cells as well as the compartmentalized pathways of sphingolipid metabolism.
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Project description:1. The effect of overloading of hamster fibroblast lysosomes with sucrose on the turnover of lysosomal and Golgi-apparatus enzymes was studied. Arylsulphatase B and UDP-galactose-N-acetylglucosamine galactosyltransferase were chosen as appropriate marker enzymes. The relative contributions of changes in the rates of synthesis and degradation to the increased activities of these enzymes after uptake of sucrose were examined by isotopic-labelling experiments. The effects of sucrose uptake on the degradation of total cellular protein and of the cytoplasmic enzyme, alkaline ribonuclease, were determined for comparative purposes. 2. The rates of enzyme synthesis in the presence and absence of sucrose were compared by pulse-labelling the cells with 14C-labelled amino acids, followed by isolation of purified enzymes and determination of their radioactivity. Sucrose uptake produced increases of 270% and 90% respectively in the rates of synthesis of arylsulphatase B and galactosyltransferase, whereas the rate of synthesis of alkaline ribonuclease was not affected. 3. The rates of degration of the enzymes were estimated by measuring the decay with time of the radioactivity of purified enzymes from prelabelled cells. In the absence of sucrose, the apparent half-lives of arylsulphatase B and galactosyltransferase were about 30 days and 40h respectively. After uptake of sucrose, the half-life of arylsulphatase B decreased to about 10 days and that of galactosyltransferase to 10h. Neither the half-life of alkaline ribonuclease (4 days) nor the rate of degradation of total cellular protein was affected by the uptake of sucrose. 4. These results indicate that the hyperactivity of lysosmal and Golgi-apparatus enzymes after the uptake of sucrose is accompanied by increases in the rates of both synthesis and degradation, and that the increased rates of degradation are insufficient to prevent accumulation of the excess of enzymes synthesized; also that the effects of sucrose uptake are restricted to the vacuolar apparatus.
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Project description:LTX-401 is an oncolytic amino acid derivative with potential immunogenic properties. Here, we demonstrate that LTX-401 selectively destroys the structure of the Golgi apparatus, as determined by means of ultrastructural analyses and fluorescence microscopic observation of cells expressing Golgi-targeted GFP reporters. Subcellular fractionation followed by mass spectrometric detection revealed that LTX-401 selectively enriched in the Golgi rather than in mitochondria or in the cytosol. The Golgi-dissociating agent Brefeldin A (BFA) reduced cell killing by LTX-401 as it partially inhibited LTX-401-induced mitochondrial release of cytochrome c and the activation of BAX. The cytotoxic effect of LTX-401 was attenuated by the double knockout of BAX and BAK, as well as the mitophagy-enforced depletion of mitochondria, yet was refractory to caspase inhibition. LTX-401 induced all major hallmarks of immunogenic cell death detectable with biosensor cell lines including calreticulin exposure, ATP release, HMGB1 exodus and a type-1 interferon response. Moreover, LTX-401-treated tumors manifested a strong lymphoid infiltration. Altogether these results support the contention that LTX-401 can stimulate immunogenic cell death through a pathway in which Golgi-localized LTX-401 operates upstream of mitochondrial membrane permeabilization.
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