Molecular characterization and substrate identification of malaria kinase PfCDPK5

描述（由申请人提供）：疟疾是全世界五岁以下儿童死亡的主要原因之一，其中大多数死亡是由恶性疟原虫感染引起的。对现有抗疟疾药物的耐药性是一个紧迫的问题，可能会阻碍有效的根除战略。对恶性疟原虫生命周期的分子理解将有助于新疗法的合理设计。P的有效出口。 从受感染的人红细胞中分离恶性疟原虫是寄生虫生命周期中的基本步骤，并且是当前抗疟疾疗法不靶向的步骤。PfCDPK5最近被鉴定为对寄生虫排出至关重要的激酶。使用最先进的 诱导蛋白质去稳定系统，这个重要基因的功能评价现在是可能的。在PfCDPK5中具有可诱导敲低的转基因寄生虫在外出之前被捕获。调节PfCDPK5水平的能力为研究这种必需激酶的激活机制和下游效应物提供了独特的资源。我们假设PfCDPK5是钙基外出信号通路的中心介导物。该提案的直接目标是获得PfCDPK5激活的分子理解，并表征其在出口信号通路中的作用。在第一个目标中， PfCDPK5激活的遗传分析将使用允许必需蛋白水平的诱导性调节的转基因寄生虫进行。在第二个目标中，PfCDPK5的下游底物将使用候选基因方法以及先进的蛋白质组学技术来发现。这些研究的长期目标和公共卫生意义是为新的抗疟疾疗法确定新的靶点。这一长期目标将作为恶性疟原虫中必需PfCDPK5信号通路的分子表征的直接结果而实现。

DESCRIPTION (provided by applicant): Malaria is among the leading causes of mortality for children under five years of age worldwide, with most of these deaths resulting from Plasmodium falciparum infection. Resistance to existing anti-malarial medications is an urgent problem and may prevent effective eradication strategies. A molecular understanding of the life cycle of P. falciparum will facilitate the rational design of new therapies. Efficient egress of P. falciparum out of an infected human red blood cell is a fundamental step in the parasite life cycle, and a step that is not targeted by current anti-malarial therapeutics. PfCDPK5 has recently been identified as a kinase that is critical for parasite egress. Using a state-of-the-art inducible protein destabilization system, functional evaluation of this essential gene is now possible. Transgenic parasites with an inducible knockdown in PfCDPK5 are arrested prior to egress. The ability to regulate the level of PfCDPK5 provides a unique resource to study both the mechanism of activation and the downstream effectors of this essential kinase. We hypothesize that PfCDPK5 is a central mediator of the calcium-based egress signaling pathway. The immediate goals of this proposal are to gain a molecular understanding of PfCDPK5 activation and characterize its role in the egress signaling pathway. In the first aim, a molecular genetic analysis of PfCDPK5 activation will be conducted using transgenic parasites that allow inducible regulation of essential protein levels. In the second aim, the downstream substrate(s) of PfCDPK5 will be discovered using both a candidate gene approach as well as advanced proteomic techniques. The long- term objectives and public health implications of these studies are to identify novel targets for new anti- malarial therapeutics. This long-term goal will be achieved as a direct result from the molecular characterization of the essential PfCDPK5 signaling pathway in P. falciparum parasites.