Malaria Parasitology Laboratory, The Francis Crick Institute, London, United Kingdom.
Mass Spectrometry Proteomics, The Francis Crick Institute, London, United Kingdom.
PLoS Pathog. 2020 Jun 22;16(6):e1008640. doi: 10.1371/journal.ppat.1008640. eCollection 2020 Jun.
Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intraerythrocytic parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, suggesting a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the apparent extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. Nuclear division and formation of intracellular structures was interrupted. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites.
泛素化是真核蛋白的一种常见翻译后修饰，在人类疟原虫（Plasmodium falciparum，Pf）中，从细胞内裂殖体到无性血期循环中的细胞外配子体阶段的过渡过程中，整体泛素化水平增加。在这里，我们在三个细胞内寄生虫阶段和细胞外配子体以及 546 种蛋白质中的 1464 个蛋白质中鉴定了特定的蛋白质底物泛素化位点（通过 ProteomeXchange 以标识符 PXD014998 提供的数据）。与前一个细胞内裂殖体阶段相比，在配子体中鉴定出 469 个泛素化蛋白，而在其中仅鉴定出 160 个，这表明与配子体成熟相关的蛋白泛素化水平大幅增加。在配子体侵入红细胞后，在第一个细胞内环阶段检测到的泛素化蛋白很少，但随着寄生虫从滋养体发育到裂殖体阶段，泛素化的明显程度增加。我们在特定的细胞功能区域中鉴定了常用的泛素化基序和泛素化蛋白组，例如参与红细胞入侵的配子体皮层蛋白、输出蛋白和组蛋白。为了研究泛素化的重要性，我们在寄生虫生长测定中筛选了泛素途径抑制剂，并发现泛素激活酶（UBA1 或 E1）抑制剂 MLN7243（TAK-243）特别有效。该小分子被证明是重组 PfUBA1 的有效抑制剂，并且 MLN7243 与寄生虫酶结合的结构同源模型突出了开发针对 Pf 特异性抑制剂的途径。我们通过雷帕霉素诱导功能性缺失 uba1 构建了一个遗传修饰的寄生虫；向重组寄生虫系中添加 MLN7243 或雷帕霉素会产生相同的表型，寄生虫的发育在裂殖体阶段被阻断。核分裂和细胞内结构的形成被打断。这些结果表明，MLN7243 的细胞内靶标是 UBA1，并且这种活性对于裂殖体最终分化为配子体至关重要。
