Biology subjects

Tascher, G.

Publications and source records attributed to Tascher, G..

3 recordsLinked to original sources

bioRxiv2021

Protein content and lipid profiling of isolated native autophagosomes

Autophagy is a central eukaryotic catabolic pathway responsible for clearance and recycling of an extensive portfolio of cargoes, which are packed in vesicles, called autophagosomes, and are delivered to lysosomes for degradation. Besides basal autophagy, which constantly degrades cellular material, the pathway is highly responsive to several stress conditions. However, the exact protein content and phospholipid composition of autophagosomes under changing autophagy conditions remain elusive so far. Here, we introduce a FACS-based approach for isolation of native unmanipulated autophagosomes and ensure the quality of the preparations. Employing quantitative proteomics and phospholipidomics, we obtained a profound cargo and lipid profile of autophagosomes purified upon basal autophagy conditions, nutrient deprivation, and proteasome inhibition. Indeed, starvation only mildly affected the content profile, while interference with proteasome activity showed stronger effects and specifically altered autophagosome cargoes. Interestingly, the phospholipid composition of autophagosomes was unaffected by the different treatments. Thus, the novel isolation method enables purification of intact autophagosomes in large quantities and allows protein content and phospholipid profiling without the requirement of exhaustive cellular fractionation or genetic manipulation.

biochemistry↗

bioRxiv2020

Autophagy activation, lipotoxicity and lysosomal membrane permeabilization synergize to promote pimozide- and loperamide-induced glioma cell death

Increasing evidence suggests that induction of lethal autophagy carries potential significance for the treatment of glioblastoma (GBM). In continuation of previous work, we demonstrate that pimozide and loperamide trigger an ATG5- and ATG7-dependent type of cell death that is significantly inhibited with the cathepsin inhibitors E64D/Pepstatin A and the lipid ROS scavenger -tocopherol in MZ-54 GBM cells. Global proteomic analysis after treatment with both drugs also revealed an increase of proteins related to lipid and cholesterol metabolic processes. These changes were accompanied by AKT1 (AKT serine/threonine kinase 1) inhibition and a massive accumulation of cholesterol and other lipids in the lysosomal compartment, indicative of impaired lipid transport/degradation. In line with these observations, pimozide and loperamide treatment were associated with a pronounced increase of bioactive sphingolipids including ceramides, glucosylceramides and sphingoid bases measured by targeted lipidomic analysis. Furthermore, pimozide and loperamide inhibited the activity of acid sphingomyelinase (ASM), increased lipid-ROS levels and promoted induction of lysosomal membrane permeabilization (LMP), as well as release of cathepsin B into the cytosol in MZ-54 wt cells. While LMP and cell death were significantly attenuated in ATG5/7 KO cells, both events were enhanced by depletion of the lysophagy receptor VCP (valosin containing protein), supporting a pro-survival function of lysophagy under these conditions. Collectively, our data suggest that pimozide and loperamide-driven autophagy and lipotoxicity synergize to induce LMP and lysosomal cell death. The results also support the notion that simultaneous overactivation of autophagy and induction of LMP represents a promising approach for the treatment of GBM.

cancer biology↗

bioRxiv2020

Growth factor receptor signaling inhibition prevents SARS-CoV-2 replication

SARS-CoV-2 infections are rapidly spreading around the globe. The rapid development of therapies is of major importance. However, our lack of understanding of the molecular processes and host cell signaling events underlying SARS-CoV-2 infection hinder therapy development. We employed a SARS-CoV-2 infection system in permissible human cells to study signaling changes by phospho-proteomics. We identified viral protein phosphorylation and defined phosphorylation-driven host cell signaling changes upon infection. Growth factor receptor (GFR) signaling and downstream pathways were activated. Drug-protein network analyses revealed GFR signaling as key pathway targetable by approved drugs. Inhibition of GFR downstream signaling by five compounds prevented SARS-CoV-2 replication in cells, assessed by cytopathic effect, viral dsRNA production, and viral RNA release into the supernatant. This study describes host cell signaling events upon SARS-CoV-2 infection and reveals GFR signaling as central pathway essential for SARS-CoV-2 replication. It provides with novel strategies for COVID-19 treatment.

systems biology↗