Iwanaga Tatsuya, Sugi Tatsuki, Kobayashi Kyousuke, Takemae Hitoshi, Gong Haiyan, Ishiwa Akiko, Murakoshi Fumi, Recuenco Frances C, Horimoto Taisuke, Akashi Hiroomi, Kato Kentaro
Department of Veterinary Microbiology, Faculty of Agriculture, The University of Tokyo, Tokyo, Japan.
Parasitol Int. 2013 Oct;62(5):423-30. doi: 10.1016/j.parint.2013.05.003. Epub 2013 May 18.
The cell cycle of Plasmodium is unique among major eukaryotic cell cycle models. Cyclin-dependent kinases (CDKs) are thought to be the key molecular switches that regulate cell cycle progression in the parasite. However, little information is available about Plasmodium CDKs. The present study was performed to investigate the effects of a CDK inhibitor, olomoucine, on the erythrocytic growth of Plasmodium falciparum. This agent inhibited the growth of the parasite at the trophozoite/schizont stage. Furthermore, we characterized the Plasmodium CDK homolog, P. falciparum cdc2-related kinase-1 (Pfcrk-1), which is a potential target of olomoucine. We synthesized a functional kinase domain of Pfcrk-1 as a GST fusion protein using a wheat germ protein expression system, and examined its phosphorylation activity. The activity of this catalytic domain was higher than that of GST-GFP control, but the same as that of a kinase-negative mutant of Pfcrk-1. After the phosphatase treatment, the labeling of [γ-(32)P]ATP was abolished. Recombinant human cyclin proteins were added to these kinase reactions, but there were no differences in activity. This report provides important information for the future investigation of Plasmodium CDKs.
疟原虫的细胞周期在主要真核细胞周期模型中独具特色。细胞周期蛋白依赖性激酶（CDKs）被认为是调节该寄生虫细胞周期进程的关键分子开关。然而，关于疟原虫CDKs的信息却很少。本研究旨在探究CDK抑制剂olomoucine对恶性疟原虫红细胞内生长的影响。该药物在滋养体/裂殖体阶段抑制了寄生虫的生长。此外，我们对疟原虫CDK同源物，即恶性疟原虫细胞分裂周期蛋白2相关激酶-1（Pfcrk-1）进行了表征，它是olomoucine的潜在作用靶点。我们使用小麦胚蛋白表达系统合成了作为GST融合蛋白的Pfcrk-1功能性激酶结构域，并检测了其磷酸化活性。该催化结构域的活性高于GST-GFP对照，但与Pfcrk-1的激酶阴性突变体相同。经过磷酸酶处理后，[γ-(32)P]ATP的标记被消除。将重组人细胞周期蛋白添加到这些激酶反应中，但活性没有差异。本报告为未来疟原虫CDKs的研究提供了重要信息。
