Characterization of a Toxoplasma gondii calcium calmodulin-dependent protein kinase homolog.
Characterization of a Toxoplasma gondii calcium calmodulin-dependent protein kinase homolog.
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DOI：
10.1186/s13071-016-1676-1
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发表时间：
2016-07-21
影响因子：
3.2
通讯作者：
Akashi H
中科院分区：
文献类型：
--
作者：
Kato K;Sugi T;Takemae H;Takano R;Gong H;Ishiwa A;Horimoto T;Akashi H
刚地弓形虫是顶复门的一种专性细胞内寄生虫，是动物和免疫功能低下的人的主要病原体，引起脑炎。了解速殖子入侵机制对于发现新的药物靶点非常重要，并可作为研究其他顶复门寄生虫的模型。我们以前表明，恶性疟原虫表达的同源物的人钙钙调蛋白依赖性蛋白激酶（CaMK），这是重要的宿主细胞入侵。在这项研究中，为了鉴定治疗弓形虫感染（另一种顶复门寄生虫）的新靶点，我们试图鉴定弓形虫中的CaMK样蛋白。弓形虫基因组，并表征其在这种寄生虫的生命周期中的作用。采用体外激酶活性测定方法，研究了一种新的类CaMK蛋白在T. gondii通过使用具有不同浓度的钙、钙调素拮抗剂或弓形虫的纯化蛋白质。弓形虫滑体蛋白。用抗该蛋白的抗体和T.刚地。我们在T. gondii、弓形虫T.弓形虫CaMK相关激酶（TgCaMKrk），其表现出钙调素非依赖性自磷酸化和底物磷酸化活性。然而，钙调素拮抗剂对其激酶活性没有影响。于T.感染gondii的细胞，TgCaMKrk定位于细胞外和细胞内速殖子的顶端。TgCaMKrk在体外磷酸化TgGAP 45用于磷酸化。我们的数据提高了我们对T.弓形虫运动和感染，寄生虫蛋白激酶和滑体之间的相互作用，以及原生动物疾病的药物靶点。
Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa and a major pathogen of animals and immunocompromised humans, in whom it causes encephalitis. Understanding the mechanism of tachyzoite invasion is important for the discovery of new drug targets and may serve as a model for the study of other apicomplexan parasites. We previously showed that Plasmodium falciparum expresses a homolog of human calcium calmodulin-dependent protein kinase (CaMK) that is important for host cell invasion. In this study, to identify novel targets for the treatment of Toxoplasma gondii infection (another apicomplexan parasite), we sought to identify a CaMK-like protein in the T. gondii genome and to characterize its role in the life-cycle of this parasite. An in vitro kinase assay was performed to assess the phosphorylation activities of a novel CaMK-like protein in T. gondii by using purified proteins with various concentrations of calcium, calmodulin antagonists, or T. gondii glideosome proteins. Indirect immunofluorescence microscopy was performed to detect the localization of this protein kinase by using the antibodies against this protein and organellar maker proteins of T. gondii. We identified a novel CaMK homolog in T. gondii, T. gondii CaMK-related kinase (TgCaMKrk), which exhibits calmodulin-independent autophosphorylation and substrate phosphorylation activity. However, calmodulin antagonists had no effect on its kinase activity. In T. gondii-infected cells, TgCaMKrk localized to the apical ends of extracellular and intracellular tachyzoites. TgCaMKrk phosphorylated TgGAP45 for phosphorylation in vitro. Our data improve our understanding of T. gondii motility and infection, the interaction between parasite protein kinases and glideosomes, and drug targets for protozoan diseases.