钙依赖蛋白激酶在柔嫩艾美耳球虫钙离子信号通路中的功能分析

Calcium is an important second messenger in a wide variety of cellular and physiological processes in all eukaryotic cells. In apicomplexan, calcium controls a number of critical events in the complex life cycles including secretion of adhesins, gliding motility, cell invasion and egress and development. Transient calcium has an important function in signal transduction networks. However, calcium influx in the cell is typically very short-lived, therefore calcium-binding proteins must be highly efficient in transmitting this signal to other enzymes. Among these calcium-binding proteins, calcium dependent protein kinases（CDPKs）are special calcium-binding serine/threonine protein kinases found only in plants, green algae, ciliates, and apicomplexan parasites. They are different from calmodulin-dependent protein kinase and protein kinase C, which play central roles in calcium signaling in mammals. Recent researches have revealed the function of several CDPKs in key parasitic life cycles, including Toxoplasmsa gondii and Plasmodium. CDPKs likely control calcium-dependent processes such as microneme secretion, cytoskeletal dynamics and regulation of motor complexes, hence influence gliding motility, cell invasion and egress and regulate developmental transitions. Since no members of the CDPK family have been found in the animal hosts, these enzymes could be suitable targets for the development of novel antiparasitic chemotherapeutics. Only limited studies have been performed in Eimeria CDPKs till now and the function of CDPKs in Eimeria Ca2+ signaling pathway still remains unknown..Eimeria tenella is one of the most important species causing avian coccidiosis and used as a model species to study Eimeria spp. In our previous report，a full length cDNA of calcium-dependent protein kinase3(EtCDPK3) of E.tenella was obtained. Results indicated that EtCDPK3 may be involved in invasion and survival of the parasite intracellular stages. Two yeast two-hybrid cDNA libraries of sporozoites and merozoites of E.tenella have been constructed and a monoclonal antibody against EtCDPK3 has also been produced. In this study, phosphorylated proteins of EtCDPK3 in the presence of calcium will be analyzed and identified by Co-immunoprecipitation and yeast two-hybrid system. Some specific inhibitors including compounds and small peptides will be selected according EtCDPK3 molecular structure. The functions of EtCDPK3 and phosphorylated substrates will be analyzed in the presence of calcium or specific inhibitors by immunolocalization and invasion inhibition assay. These results will provide important rationale to understand the molecular mechanism of EtCDPK3 in E.tenella calcium signaling pathways and find new targets for development of new vaccines and drugs against coccidiosis.

Ca2+作为第二信使在顶复器原虫生活史中起关键作用，其能否快速准确传递信号取决于与之结合的效应分子。钙依赖蛋白激酶(CDPKs)仅在植物、绿藻、纤毛虫和顶复器原虫中发现，已证实CDPKs是顶复器原虫最重要的Ca2+效应分子，调控着弓形虫和疟原虫的运动、入侵、逃逸等关键生理过程，是研究顶复器原虫病防治新方法的重要靶标。迄今CDPKs在鸡球虫中的功能尚不清楚。本项目拟在已获得柔嫩艾美耳球虫CDPK3(EtCDPK3)单克隆抗体以及子孢子和裂殖子酵母cDNA文库的基础上，采用CoIP和酵母双杂交技术捕获EtCDPK3磷酸化底物，筛选特异抑制剂，分析Ca2+和抑制剂对EtCDPK3和磷酸化底物在转录和翻译水平、虫体入侵和逃逸等方面的影响；用体外抑制和免疫共定位等技术研究EtCDPK3在球虫Ca2+信号通路中发挥功能的分子机制，为寻找防治鸡球虫病重要关键分子奠定基础，具有重要科学意义和潜在应用价值。

Ca2+作为第二信史在顶复器原虫生活史中起关键作用，钙依赖蛋白激酶（CDPKs）是顶复器原虫Ca2+信号通路中最重要的效应分子，仅在植物、绿藻、纤毛虫及顶复器原虫发现，调控着弓形虫和疟原虫的运动、入侵、逃逸等关键生理过程，是研究顶复器原虫病防治的重要靶标。迄今，CDPKs在鸡球虫中的功能尚不清楚。本研究对柔嫩艾美耳球虫CDPK4（EtCDPK4）特性和功能进行了研究。利用RACE获得EtCDPK4全长cDNA序列，分析显示该蛋白具有CDPKs家族成员特征性保守结构域。研究显示EtCDPK4在E. tenella子孢子表达最高，主要位于子孢子和第二代裂殖子前端表面，在虫体入侵宿主细胞过程中发挥重要功能。EtCDPK4酶催化活性随着Ca2+浓度升高而增大。筛选获得4种EtCDPK4特异性抑制剂（W-7、H-7、H-89和Myristoylated Peptide），能有效抑制子孢子入侵DF-1细胞。利用酵母双杂交和Co-IP等筛选获得了12个EtCDPK4潜在互作蛋白，利用免疫共定位和BiFC等对6个蛋白（丝氨酸蛋白酶抑制剂（EtSerpin），DNA介导的RNA聚合酶Ⅰ和Ⅲ亚基（EtRPAC1），14-3-3蛋白（Et14-3-3），延长因子1α（EtEF1α）、翻译起始因子eIF-5A（EteIF-5A）、5-吡咯啉羧酸还原酶（EtPYCR）进行了验证，发现3个蛋白（EtSerpin，Et14-3-3，EteIF-5A）与EtCDPK4存在互作关系。同时筛选获得17个EtCDPK3潜在的互作蛋白，对五个蛋白（2个未知蛋白EtCHP12，EtCHP3、锌指蛋白EtAN1-ZnFP、延长因子1αEtEF1α、二磷酸核苷激酶（EtNDK））进行验证，结果这几个蛋白与EtCDPK3都不互作。对6个潜在互作蛋白（Et14-3-3，EtEF1α，EteIF-5A，EtPYCR，EtCHP3和EtZN1-ZnFP）特性研究发现，蛋白都在虫体不同发育阶段差异表达；EtEF1α和EteIF-5A位于子孢子顶端和裂殖子整个细胞质。EtPYCR和 Et14-3-3位于子孢子顶端和表面以及裂殖子表面；EtCHP3位于子孢子和裂殖子表面；EtAN1-ZnFP均匀分布在子孢子细胞质。除Et14-3-3，其余5个蛋白都参与了子孢子入侵宿主细胞。这些结果为鸡球虫病防治新方法提供分子靶标。