Specificity of Plasmodium falciparum protein export

疟疾寄生虫恶性疟原虫出口几百个 蛋白质进入其宿主细胞，以调节营养摄取，感染RBC生物物理 特性、对血管内皮的粘附和宿主免疫应答。这些 输出的蛋白质效应物在寄生虫ER中合成，穿过 分泌系统，然后从寄生泡（PV） 进入宿主红细胞大多数都有一个蛋白质输出元件（PEXEL）， 但被天冬氨酸蛋白酶协同切割 plasmepsin V.加工后的蛋白质如何被识别用于出口根本不清楚。 含PEXEL报告基因的成熟N-末端中的某些突变 构建体可以阻止输出，但将成熟的N-末端改变为所有丙氨酸 并不影响出口。我们假设出口是 分泌蛋白和特定突变阻断输出或促进PV滞留。 我们认为这些突变决定了伴侣的识别。建立这一 模型，我们必须确定伴侣（S）赋予特异性。 为了解决这些问题，Aim 1将专注于蛋白质机制， 中介出口特异性。出口目的地和PV保留蛋白质将被拉下 从肺静脉和急诊室比较蛋白质组学分析将是 用于确定出口途径的关键组成部分。邻近生物素化 这将是第二种分析方法。候选相互作用蛋白 将在相互下拉中得到验证。这将为未来的逆转铺平道路 遗传和生化评估。我们预计， 将对蛋白质输出的机制产生深刻的见解， 疟疾的发病机制我们希望这项工作将为新的 治疗这种毁灭性疾病的方法。

The malaria parasite Plasmodium falciparum exports several hundred proteins into its host cell to modulate nutrient uptake, infected RBC biophysical properties, adherence to vascular endothelium and host immune response. These exported protein effectors are synthesized in the parasite ER, traverse the secretory system and are then translocated from the parasitophorous vacuole (PV) into the host RBC. Most have a protein export element (PEXEL) that is essential for the export process but is cleaved co-translationally by the aspartic protease plasmepsin V. How the processed protein is recognized for export is not at all clear. Certain mutations in the mature N-terminus of PEXEL-containing reporter constructs can prevent export, but changing the mature N-terminus to all alanines does not impair export. We hypothesize that export is the default pathway for secretory proteins and that specific mutations block export or promote PV retention. We posit that these mutations dictate recognition by a chaperone. To establish this model, we must identify the chaperone(s) that confer specificity. To address these questions, Aim 1 will focus on the protein machinery that mediates export specificity. Export-destined and PV retained proteins will be pulled down from the PV and from the ER. Comparative proteomic analysis will be employed to identify key components of the export pathway. Proximity biotinylation will comprise a second approach to this analysis. Candidate interacting proteins will be validated in reciprocal pulldowns. This will pave the way for future reverse genetic and biochemical assessments. We anticipate that the proposed studies will yield great insight into the mechanism of protein export that is so important to the pathogenesis of malaria. We hope that this work will point the way to new therapies for this devastating disease.