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Reversibility of membrane N-glycome of HeLa cells upon treatment with epigenetic inhibitors.

ABSTRACT: Glycans are essential regulators of protein function and are now in the focus of research in many physiological and pathophysiological processes. There are numerous modes of regulating their biosynthesis, including epigenetic mechanisms implicated in the expression of glyco-genes. Since N-glycans located at the cell membrane define intercellular communication as well as a cellular response to a given environment, we developed a method to preferentially analyze this fraction of glycans. The method is based on incorporation of living cells into polyacrylamide gels, partial denaturation of membrane proteins with 3 M urea and subsequent release of N-glycans with PNGase F followed by HPLC analysis. Using this newly developed method, we revealed multiple effects of epigenetic inhibitors Trichostatin A, sodium butyrate and zebularine on the composition of N-glycans in human cells. The induced changes were found to be reversible after inhibitor removal. Given that many epigenetic inhibitors are currently explored as a therapeutic strategy in treatment of cancer, wherein surface glycans play an important role, the presented work contributes to our understanding of their efficiency in altering the N-glycan profile of cancer cells in culture.

SUBMITTER: Horvat T

PROVIDER: S-EPMC3545996 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Mitochondria are the main bioenergetic organelles of cells. Exposure to chemicals targeting mitochondria therefore generally results in the development of toxicity. The cellular response to perturbations in cellular energy production is a balance between adaptation, by reorganisation and organelle biogenesis, and sacrifice, in the form of cell death. In homeostatic conditions, aerobic mitochondrial energy production requires the maintenance of a mitochondrial membrane potential (MMP). Chemicals can perturb this MMP, and the extent of this perturbation depends both on the pharmacokinetics of the chemicals and on downstream MMP dynamics. Here we obtain a quantitative understanding of mitochondrial adaptation upon exposure to various mitochondrial respiration inhibitors by applying mathematical modeling to partially published high-content imaging time-lapse confocal imaging data, focusing on MMP dynamics in HepG2 cells over a period of 24 h. The MMP was perturbed using a set of 24 compounds, either acting as uncoupler or as mitochondrial complex inhibitor targeting complex I, II, III or V. To characterize the effect of chemical exposure on MMP dynamics, we adapted an existing differential equation model and fitted this model to the observed MMP dynamics. Complex III inhibitor data were better described by the model than complex I data. Incorporation of pharmacokinetic decay into the model was required to obtain a proper fit for the uncoupler FCCP. Furthermore, oligomycin (complex V inhibitor) model fits were improved by either combining pharmacokinetic (PK) decay and ion leakage or a concentration-dependent decay. Subsequent mass spectrometry measurements showed that FCCP had a significant decay in its PK profile as predicted by the model. Moreover, the measured oligomycin PK profile exhibited only a limited decay at high concentration, whereas at low concentrations the compound remained below the detection limit within cells. This is consistent with the hypothesis that oligomycin exhibits a concentration-dependent decay, yet awaits further experimental verification with more sensitive detection methods. Overall, we show that there is a complex interplay between PK and MMP dynamics within mitochondria and that data-driven modeling is a powerful combination to unravel such complexity.

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Reversibility of membrane N-glycome of HeLa cells upon treatment with epigenetic inhibitors.

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