Functional analysis of the "Apical Membrane Antigen 1": Investigating the role of phosphorylation of the blood-stage vaccine candidate in the malaria parasite Plasmodium falciparum

疟疾寄生虫恶性疟原虫生命周期中的一个关键过程是侵入人类红细胞。进入宿主细胞需要顶端膜抗原1（AMA-1），一种位于裂殖子微线中的I型跨膜蛋白。虽然AMA-1正在发展成为领先的血液阶段疟疾疫苗候选者，但其在入侵中的确切作用仍不清楚。对AMA-1胞质结构域的突变分析表明，磷酸化在介导宿主细胞侵袭中起重要作用。建议深入验证I型跨膜蛋白的这种磷酸化作为巨噬细胞侵袭过程中的一个关键步骤。首先，将阐明AMA-1磷酸化的精确时间及其功能后果。其次，PfPKA，一种已知能够磷酸化重组AMA-1的激酶，将使用激酶特异性抑制文库在体内和体外验证为推定的药物靶标。将尝试鉴定参与磷酸化事件的其他激酶。最后，将分析已知参与入侵的其他1型跨膜蛋白（如DBL，RH和TRAP家族）的假定磷酸化，并研究其功能后果。靶向负责的激酶为新的治疗药物策略提供了有吸引力的靶标，其独立于基于抗体的方法，所述基于抗体的方法受到疟疾寄生虫中粘附素的多态性细胞外结构域中的抗原逃逸区的阻碍。

A key process in the lifecycle of the malaria parasite Plasmodium falciparum is the invasion of human erythrocytes. Entry into the host cell requires the apical membrane antigen 1 (AMA-1), a type I transmembrane protein located in the micronemes of the merozoite. Although AMA-1 is evolving into the leading blood-stage malaria vaccine candidate, its precise role in invasion is still unclear. Mutational analysis of the cytoplasmic domain of AMA-1 suggested an important role for the phosphorylation to mediate host cell invasion. It is proposed to validate in depth this phosphorylation of type I transmembrane proteins as one key step in the invasion process of erythocytes. Firstly, the precise timing of AMA-1 phosphorylation and its functional consequences will be elucidated. Secondly, PfPKA, a kinase known to be capable to phosphorylate recombinant AMA-1, will be validated in vivo and in vitro as a putative drug target using a kinase specific inhibitory library. Additional kinases involved in the phosphorylation event will be attempted to identify. Finally, the putative phosphorylation of other type 1 transmembrane proteins known to be involved in invasion (like DBL, RH and the TRAP family) will be analyzed and the functional consequences investigated. Targeting the responsible kinase(s) provide an attractive target for novel therapeutic drug strategies independent of an antibody based approach that is hampered by antigenic escape regions in polymorphic extracellular domains of adhesins in the malaria parasite.