Molecular basis of effector protein export in the malaria parasite Plasmodium falciparum

疟原虫恶性疟原虫效应蛋白输出的分子基础

• 批准号: 10260440
• 负责人: Chi-Min Ho
• 金额: $ 40.5万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-10 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10260440
• 关键词: Academia; Affect; Antimalarials; Award; Biochemical; Biochemistry; Biological Assay; Biophysics; Blood; Bypass; Cell membrane; Cells; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Communities; Contracts; Cryo-electron tomography; Cryoelectron Microscopy; Cytosol; Development; Disease; Doctor of Philosophy; Drug Design; Drug Targeting; Drug resistance; Epitopes; Equipment; Erythrocytes; Faculty; Fostering; Funding; Future; Gatekeeping; Goals; Human; Immune system; Immunology; In Situ; In Vitro; Industry; Infrastructure; Interdisciplinary Study; Ions; Life Cycle Stages; Malaria; Mediating; Medicine; Membrane; Membrane Proteins; Mentors; Metabolic; Methods; Microbiology; Modification; Molecular; Nutrient; Parasite resistance; Parasites; Parasitic Diseases; Pathogenesis; Pathway interactions; Plasma Cells; Plasmodium; Plasmodium falciparum; Populations at Risk; Positioning Attribute; Process; Protein Biochemistry; Protein Export Pathway; Protein Subunits; Protein translocation; Proteins; Proteome; Recombinants; Regulation; Research; Resolution; Role; Scientist; Source; Structure; System; Techniques; Therapeutic; Training; Universities; Vacuole; Vision; Visualization; Work; World Health Organization; base; biological systems; collaborative environment; design; drug discovery; experience; inhibitor/antagonist; insight; member; multidisciplinary; novel; novel therapeutics; particle; pathogen; professor; programs; protein complex; screening; square foot; tenure track; wasting

Project Summary Malaria is a devastating parasitic disease that affects more than 200 million people annually, resulting in nearly 500,000 deaths each year. As of 2018, the World Health Organization estimates that 3.8 billion people, roughly half the world's population, are at risk of contracting malaria, and the rise of drug-resistant parasites has created a desperate need for new anti-malarial drugs. While most intracellular pathogens export a limited repertoire of effector proteins to co-opt existing host-cell metabolic machineries, the malaria-causing parasite Plasmodium falciparum exports more than 10% of its proteome into its host, the human red blood cell, during the blood stages of its life cycle. The hundreds of proteins in the P. falciparum exportome extensively remodel host erythrocytes, creating the infrastructure needed to import nutrients, export waste, and evade the host immune system. The export of these hundreds of proteins is complicated by the fact that the malaria parasite conceals itself inside a parasitophorous vacuole (PV) derived from invagination of the host cell plasma membrane during invasion. Following secretion into the PV, proteins destined for export must be unfolded and transported across the PV membrane (PVM) into the host cell in an ATP-dependent process. The export pathway is essential for parasite survival, making members of the pathway attractive potential drug targets. The complexity and breadth of its host-cell remodeling machinery make P. falciparum a rich and exciting system for the study of host-pathogen interactions. However, many of the molecular mechanisms underlying this parasite's ability to hijack human red blood cells remain enigmatic, as much of the P. falciparum proteome has proven recalcitrant to structural and biochemical characterization using traditional recombinant approaches. The goal of the proposed work is to leverage and build upon the latest advances in single-particle cryo electron microscopy and cryo focused ion beam-enabled in situ cryo electron tomography to elucidate the molecular mechanisms underlying effector protein export in P. falciparum and to identify promising targets for structure-based design of new anti-malarial therapeutics. Three aims are proposed to accomplish these goals: 1) Establish an in vitro translocation activity assay for the Plasmodium Translocon of Exported Proteins (PTEX), a novel and essential membrane protein complex, through which all exported effector proteins must pass in order to reach the host cell cytosol. The established assay will enable biochemical characterization of the molecular mechanism of protein translocation and screening of inhibitors obtained via structure-guided design of PTEX inhibitors. 2) Structure determination of novel protein complexes of the P. falciparum exportome. 3) Direct visualization of the supramolecular effector protein export machinery in situ at the host-pathogen interface in P. falciparum-infected erythrocytes. The proposed work will provide insight into the pathogenesis of this deadly disease, identify new malarial drug targets, and enable structure-guided design of novel anti-malarial therapeutics.

项目概要 疟疾是一种毁灭性的寄生虫病，每年影响超过 2 亿人，导致近 每年有 50 万人死亡。截至 2018 年，世界卫生组织估计有 38 亿人，大约 世界上一半的人口面临感染疟疾的风险，抗药性寄生虫的增加导致 迫切需要新的抗疟疾药物。虽然大多数细胞内病原体输出有限的库 效应蛋白来选择现有的宿主细胞代谢机制，即引起疟疾的寄生虫疟原虫 在血液阶段，恶性疟原虫将超过 10% 的蛋白质组输出到其宿主——人类红细胞中 其生命周期。恶性疟原虫输出组中的数百种蛋白质广泛重塑宿主红细胞， 建立输入营养物质、输出废物和逃避宿主免疫系统所需的基础设施。这 由于疟疾寄生虫将自身隐藏在体内，因此这数百种蛋白质的输出变得复杂。 寄生液泡（PV）源自入侵过程中宿主细胞质膜的内陷。 分泌到 PV 后，用于输出的蛋白质必须展开并穿过 PV 膜（PVM）通过 ATP 依赖性过程进入宿主细胞。输出途径对于寄生虫至关重要 生存，使该途径的成员有吸引力的潜在药物靶点。其复杂性和广度 宿主细胞重塑机制使恶性疟原虫成为研究宿主病原体的丰富且令人兴奋的系统 互动。然而，这种寄生虫劫持人类红色能力的许多分子机制 血细胞仍然是个谜，因为许多恶性疟原虫蛋白质组已被证明难以适应结构和 使用传统重组方法进行生化表征。拟议工作的目标是 利用并建立在单粒子冷冻电子显微镜和冷冻聚焦离子方面的最新进展 束原位冷冻电子断层扫描阐明效应器的分子机制 恶性疟原虫中的蛋白质输出，并确定基于结构的新型抗疟疾药物设计的有希望的目标 疗法。为了实现这些目标，提出了三个目标：1）建立体外易位活动 疟原虫输出蛋白易位子 (PTEX) 的测定，这是一种新型且重要的膜蛋白 复合体，所有输出的效应蛋白必须通过该复合体才能到达宿主细胞胞浆。这 建立的检测方法将能够对蛋白质易位的分子机制进行生化表征 以及通过 PTEX 抑制剂的结构指导设计获得的抑制剂的筛选。 2) 结构测定 恶性疟原虫输出组的新型蛋白质复合物。 3）超分子效应器的直接可视化 恶性疟原虫感染的红细胞中宿主-病原体界面处的蛋白质输出机制。这 拟议的工作将深入了解这种致命疾病的发病机制，确定新的疟疾药物靶标， 并实现新型抗疟疾疗法的结构引导设计。