Specificity of Plasmodium falciparum protein export

恶性疟原虫蛋白输出的特异性

• 批准号: 10632093
• 负责人: Daniel E. Goldberg
• 金额: $ 19.46万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10632093
• 关键词: Address; Adherence; Africa South of the Sahara; Alanine; Aspartic Endopeptidases; Biochemical; Biotinylation; Brefeldin A; Cells; Child; Development; Disease; Elements; Erythrocytes; Future; Immune response; Immunoprecipitation; Impairment; Interruption; Investigation; Malaria; Mass Spectrum Analysis; Mediating; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Mutation; Nutrient; Parasites; Pathogenesis; Pathway interactions; Persons; Phenotype; Plasmodium falciparum; Process; Protein Export Pathway; Proteins; Proteomics; Reporter; Specificity; Streptavidin; System; Vacuole; Vascular Endothelium; Work; biophysical properties; comparative; experimental study; insight; knock-down; mutant; novel therapeutics; plasmepsin; prevent; reverse genetics; secretory protein; therapeutic target; uptake

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.

疟原虫恶性疟原虫出口数百种 蛋白质进入其宿主细胞以调节营养吸收，感染红细胞生物物理 特性、血管内皮粘附性和宿主免疫反应。这些 输出的蛋白质效应子在寄生虫内质网中合成，穿过 分泌系统，然后从寄生液泡（PV）转移 进入宿主红细胞。大多数都具有必需的蛋白质输出元件 (PEXEL) 用于导出过程，但被天冬氨酸蛋白酶共翻译切割 加工后的蛋白质如何被识别用于出口尚不清楚。 含有 PEXEL 的报告基因的成熟 N 末端的某些突变 构建体可以阻止输出，但将成熟的 N 末端更改为所有丙氨酸 不损害出口。我们假设出口是默认途径 分泌蛋白和特定突变阻止输出或促进 PV 保留。 我们假设这些突变决定了伴侣的识别。为了建立这个 模型中，我们必须确定赋予特异性的伴侣。 为了解决这些问题，目标 1 将重点关注蛋白质机制 调节出口特异性。用于出口和PV保留的蛋白质将被撤出 从 PV 和 ER 向下。比较蛋白质组学分析将 用于确定出口途径的关键组成部分。邻近生物素化 将包含第二种分析方法。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。我们预计拟议的研究 将深入了解蛋白质输出的机制，这对于 疟疾的发病机制。我们希望这项工作能够为新的方向指明道路 针对这种毁灭性疾病的治疗方法。