Institute of Molecular Medicine I, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, 40225, Germany.
Molecular Proteomics Laboratory, Biological Medical Research Centre, Heinrich Heine University, 40225, Düsseldorf, Germany.
Cell Commun Signal. 2023 Oct 5;21(1):275. doi: 10.1186/s12964-023-01275-1.
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.
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.
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.
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.
细菌次生代谢产物灵菌红素已被证明具有抗癌、抗疟、抗菌和免疫调节作用。在癌症方面，它已被报道能够影响癌细胞而不影响非恶性细胞，这使得灵菌红素成为抗癌药物发现的有前途的先导化合物。然而，目前尚未通过实验确定其直接的蛋白质靶标。
我们使用基于质谱的热蛋白质组谱分析来鉴定灵菌红素的靶蛋白。为了进行靶标验证，我们采用了基因敲除方法和电子显微镜。
我们确定了高尔基堆叠蛋白 GRASP55 是灵菌红素的靶蛋白。我们表明，灵菌红素处理严重影响高尔基的形态和功能，并且在 GRASP55 敲除细胞中，灵菌红素依赖性细胞毒性部分降低。我们还发现，灵菌红素处理导致组织蛋白酶活性降低和自噬流整体阻断，而自噬体标记物 LC3 和溶酶体标记物 LAMP1 的共定位明显增强。最后，我们观察到自噬体在 GRASP55 阳性结构处积累，表明高尔基功能的改变参与了这种天然化合物对自噬的抑制作用。
综上所述，我们提出灵菌红素通过调节细胞器碱化和高尔基堆叠蛋白 GRASP55 以相互关联的作用方式影响自噬和高尔基器的完整性。视频摘要。
