Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Str. 2+4, 14195 Berlin, Germany.
Institute of Chemistry and Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.
Toxins (Basel). 2024 May 10;16(5):219. doi: 10.3390/toxins16050219.
Saponin-mediated endosomal escape is a mechanism that increases the cytotoxicity of type I ribosome-inactivating proteins (type I RIPs). In order to actualize their cytotoxicity, type I RIPs must be released into the cytosol after endocytosis. Without release from the endosomes, type I RIPs are largely degraded and cannot exert their cytotoxic effects. Certain triterpene saponins are able to induce the endosomal escape of these type I RIPs, thus increasing their cytotoxicity. However, the molecular mechanism underlying the endosomal escape enhancement of type I RIPs by triterpene saponins has not been fully elucidated. In this report, we investigate the involvement of the basic amino acid residues of dianthin-30, a type I RIP isolated from the plant L., in endosomal escape enhancement using alanine scanning. Therefore, we designed 19 alanine mutants of dianthin-30. Each mutant was combined with SO1861, a triterpene saponin isolated from the roots of L., and subjected to a cytotoxicity screening in Neuro-2A cells. Cytotoxic screening revealed that dianthin-30 mutants with lysine substitutions did not impair the endosomal escape enhancement. There was one particular mutant dianthin, Arg24Ala, that exhibited significantly reduced synergistic cytotoxicity in three mammalian cell lines. However, this reduction was not based on an altered interaction with SO1861. It was, rather, due to the impaired endocytosis of dianthin Arg24Ala into the cells.
皂素介导的内体逃逸是增加 I 型核糖体失活蛋白(type I RIPs)细胞毒性的一种机制。为了实现其细胞毒性,I 型 RIP 必须在胞吞作用后从内体中释放到细胞质中。如果没有从内体中释放,I 型 RIP 就会被大量降解,无法发挥其细胞毒性作用。某些三萜皂苷能够诱导这些 I 型 RIP 的内体逃逸,从而增加其细胞毒性。然而,三萜皂苷增强 I 型 RIP 内体逃逸的分子机制尚未完全阐明。在本报告中,我们使用丙氨酸扫描研究了从植物 L.中分离出的 I 型 RIP 二蒽酮-30 的碱性氨基酸残基在促进内体逃逸中的作用。因此,我们设计了 19 个二蒽酮-30 的丙氨酸突变体。每个突变体都与从 L.根部分离出的三萜皂苷 SO1861 结合,并在 Neuro-2A 细胞中进行细胞毒性筛选。细胞毒性筛选显示,赖氨酸取代的二蒽酮-30 突变体不会损害内体逃逸增强。有一个特定的二蒽酮突变体 Arg24Ala,在三种哺乳动物细胞系中表现出显著降低的协同细胞毒性。然而,这种减少不是基于与 SO1861 的改变相互作用。相反,是由于 Arg24Ala 二蒽酮的内吞作用受损。