Department of Biophysics and Chemical Biology, Seoul National University, Seoul, 08826, Korea.
CRI Center for Chemical Proteomics, Department of Chemistry, Seoul National University, Seoul, 08826, Korea.
Commun Biol. 2021 Sep 23;4(1):1123. doi: 10.1038/s42003-021-02643-8.
The highly cytotoxic marine natural product callyspongiolide holds great promise as a warhead of antibody-drug conjugate in cancer therapeutics; however, the mechanism underlying its cytotoxicity remains unclear. To elucidate how callyspongiolide kills cells, we employed label-free target identification with thermal stability-shift-based fluorescence difference in two-dimensional (2-D) gel electrophoresis (TS-FITGE), which allowed observation of a unique phenomenon of protein-spot separation on 2-D gels upon treatment with callyspongiolide at increasing temperatures. During our exploration of what proteins were associated with this phenomenon as well as why it happens, we found that callyspongiolide induces mitochondrial/lysosomal dysfunction and autophagy inhibition. Moreover, molecular biology studies revealed that callyspongiolide causes lysosomal dysfunction, which induces cellular iron depletion and leads to mitochondrial dysfunction and subsequent cytotoxicity. Notably, these effects were rescued through iron supplementation. Although our approach was unable to reveal the direct protein targets of callyspongiolide, unique phenomena observed only by TS-FITGE provided critical insight into the mechanism of action of callyspongiolide and specifically its cytotoxic activity via induction of mitochondrial dysfunction through cellular iron depletion caused by lysosomal deacidification, which occurred independent of known programmed cell death pathways.
高细胞毒性海洋天然产物 callyspongiolide 有望成为癌症治疗中抗体药物偶联物的弹头；然而，其细胞毒性的机制仍不清楚。为了阐明 callyspongiolide 如何杀死细胞，我们采用无标记的靶标鉴定方法，即基于二维凝胶电泳（2-DGE）的热稳定性移位荧光差异（TS-FITGE），该方法可以观察到 callyspongiolide 在升高温度下处理时，蛋白质斑点在 2-D 凝胶上出现独特的分离现象。在探索与这种现象相关的蛋白质以及为什么会发生这种现象的过程中，我们发现 callyspongiolide 诱导线粒体/溶酶体功能障碍和自噬抑制。此外，分子生物学研究表明 callyspongiolide 引起溶酶体功能障碍，导致细胞内铁耗竭，进而导致线粒体功能障碍和随后的细胞毒性。值得注意的是，这些效应可以通过铁补充来挽救。虽然我们的方法无法揭示 callyspongiolide 的直接蛋白靶标，但仅通过 TS-FITGE 观察到的独特现象为 callyspongiolide 的作用机制提供了关键的见解，特别是通过溶酶体去酸化引起的细胞内铁耗竭导致的线粒体功能障碍引起的细胞毒性活性，这一过程独立于已知的程序性细胞死亡途径。
