The Golgi stacking protein GRASP55 is targeted by the natural compound prodigiosin.

BACKGROUND: The bacterial secondary metabolite prodigiosin has been shown to exert anticancer, antimalarial, antibacterial and immunomodulatory properties. With regard to cancer, it has been reported to affect cancer cells but not non-malignant cells, rendering prodigiosin a promising lead compound for anticancer drug discovery. However, a direct protein target has not yet been experimentally identified. METHODS: We used mass spectrometry-based thermal proteome profiling in order to identify target proteins of prodigiosin. For target validation, we employed a genetic knockout approach and electron microscopy. RESULTS: We identified the Golgi stacking protein GRASP55 as target protein of prodigiosin. We show that prodigiosin treatment severely affects Golgi morphology and functionality, and that prodigiosin-dependent cytotoxicity is partially reduced in GRASP55 knockout cells. We also found that prodigiosin treatment results in decreased cathepsin activity and overall blocks autophagic flux, whereas co-localization of the autophagosomal marker LC3 and the lysosomal marker LAMP1 is clearly promoted. Finally, we observed that autophagosomes accumulate at GRASP55-positive structures, pointing towards an involvement of an altered Golgi function in the autophagy-inhibitory effect of this natural compound. CONCLUSION: Taken together, we propose that prodigiosin affects autophagy and Golgi apparatus integrity in an interlinked mode of action involving the regulation of organelle alkalization and the Golgi stacking protein GRASP55. Video Abstract.

Modulation of Specific Sphingosine-1-Phosphate Receptors Augments a Repair Mediating Schwann Cell Phenotype.

Transdifferentiation of Schwann cells is essential for functional peripheral nerve regeneration after injury. By activating a repair program, Schwann cells promote functional axonal regeneration and remyelination. However, chronic denervation, aging, metabolic diseases, or chronic inflammatory processes reduce the transdifferentiation capacity and thus diminish peripheral nerve repair. It was recently described that the sphingosine-1-phosphate receptor (S1PR) agonist Fingolimod enhances the Schwann cell repair phenotype by activation of dedifferentiation markers and concomitant release of trophic factors resulting in enhanced neurite growth. Since Fingolimod targets four out of five S1PRs (S1P1, S1P3-5) possibly leading to non-specific adverse effects, identification of the main receptor(s) responsible for the observed phenotypic changes is mandatory for future specific treatment approaches. Our experiments revealed that S1P3 dominates and that along with S1P1 acts as the responsible receptor for Schwann cell transdifferentiation as revealed by the combinatory application of specific agonists and antagonists. Targeting both receptors reduced the expression of myelin-associated genes, increased PDGF-BB representing enhanced trophic factor expression likely to result from c-Jun induction. Furthermore, we demonstrated that S1P4 and S1P5 play only a minor role in the adaptation of the repair phenotype. In conclusion, modulation of S1P1 and S1P3 could be effective to enhance the Schwann cell repair phenotype and thus stimulate proper nerve repair.

Fin56-induced ferroptosis is supported by autophagy-mediated GPX4 degradation and functions synergistically with mTOR inhibition to kill bladder cancer cells.

Ferroptosis is a form of regulated cell death that emerges to be relevant for therapy-resistant and dedifferentiating cancers. Although several lines of evidence suggest that ferroptosis is a type of autophagy-dependent cell death, the underlying molecular mechanisms remain unclear. Fin56, a type 3 ferroptosis inducer, triggers ferroptosis by promoting glutathione peroxidase 4 (GPX4) protein degradation via a not fully understood pathway. Here, we determined that Fin56 induces ferroptosis and autophagy in bladder cancer cells and that Fin56-triggered ferroptosis mechanistically depends on the autophagic machinery. Furthermore, we found that autophagy inhibition at different stages attenuates Fin56-induced oxidative stress and GPX4 degradation. Moreover, we investigated the effects of Fin56 in combination with Torin 2, a potent mTOR inhibitor used to activate autophagy, on cell viability. We found that Fin56 synergizes with Torin 2 in cytotoxicity against bladder cancer cells. Collectively, our findings not only support the concept that ferroptosis is a type of autophagy-dependent cell death but imply that the combined application of ferroptosis inducers and mTOR inhibitors is a promising approach to improve therapeutic options in the treatment of bladder cancer.