Functional Analysis of Novel Components of the Toxoplasma Inner Membrane Complex

弓形虫内膜复合物新成分的功能分析

• 批准号: 10444432
• 负责人: Peter John Bradley
• 金额: $ 44.88万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10444432
• 关键词: Ablation; Acylation; Affect; Alveolar; Alveolus; Animals; Apical; Apicomplexa; Architecture; Binding; Biotinylation; C-terminal; Cell membrane; Cells; Cellular biology; Coccidiosis; Coiled-Coil Domain; Complement; Complex; Cone; Cryo-electron tomography; Cytoskeletal Filaments; Cytoskeleton; Daughter; Disease; Eimeria tenella; Elements; Falciparum Malaria; Funding; Grant; Human; Invaded; Lead; Life Style; Mediating; Medical; Membrane; Microtubules; Molecular; Motor; Names; Neospora caninum; Organelles; Parasites; Pattern; Peripheral; Play; Process; Proteins; Proteome; Reporting; Role; Series; Structure; Surgical sutures; System; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Vesicle; Work; base; cell motility; crosslink; daughter cell; design; human pathogen; in vivo; knock-down; knockout gene; member; new therapeutic target; novel; novel therapeutics; obligate intracellular parasite; pathogen; protein complex; protein function; scaffold; unnatural amino acids

PROJECT SUMMARY Toxoplasma gondii and related apicomplexan parasites contain a specialized organelle called the inner membrane complex (IMC) that plays essential roles in host cell invasion and daughter cell formation. The IMC consists of flattened membrane vesicles and a supporting cytoskeletal meshwork that is flanked by the plasma membrane and subpellicular microtubules at the periphery of the parasite. Recent in vivo biotinylation (BioID) studies have revealed a surprising level of compartmentalization within the IMC organelle, with distinct groups of proteins segregating to the cone-shaped apical cap, the body, or to the basal complex of both the cytoskeletal and membrane subcompartments. Analyses from our group and others have exposed new essential functions of the IMC including the role of the apical cap complex AC9/AC10/ERK7 in mounting the conoid which is essential for organelle secretion and invasion as well as the conserved early daughter bud protein IMC32 that is essential for replication. In this renewal, we will expand on these studies to determine precisely how these and other critical IMC components are able to carry out their functions. First, we will determine the role of novel apical cap proteins that are putative interactors of the AC9/AC10/ERK7 complex and assess their role in apical cap function and conoid assembly. We will then explore how IMC32 collaborates with the newly discovered partner IMC48 to function at the earliest stages of parasite division and use these proteins to further explore the early daughter bud proteome. Finally, we will exploit our recently developed photoreactive unnatural amino acid system as well as cryo-electron tomography to determine how critical alveolins of the parasite cytoskeleton are organized to serve as scaffolds for the organelle. Together, this project will promote a much deeper understanding of the architecture and function of the Toxoplasma IMC. As this organelle is parasite-specific and not present in its human host, determining precisely how these essential IMC components function promises to enable the design of novel therapies against T. gondii and other apicomplexan parasites.

项目摘要 弓形虫和相关的顶复门寄生虫含有一个专门的细胞器称为内 膜复合物（IMC）在宿主细胞入侵和子细胞形成中起重要作用。法团校董会 由扁平的膜囊泡和支持细胞骨架网组成，细胞骨架网的两侧是血浆 膜和膜下微管在寄生虫的外围。最近的体内生物素化（BioID） 研究已经揭示了IMC细胞器中令人惊讶的区域化水平，具有不同的组 蛋白质分离到锥形顶帽，身体，或到细胞骨架的基底复合体 和膜亚区室。我们小组和其他人的分析揭示了新的基本功能 包括顶帽复合物AC 9/AC 10/ERK 7在安装圆锥体中的作用， 细胞器分泌和入侵以及保守的早期子芽蛋白IMC 32是必不可少的 用于复制。在这次更新中，我们将扩大这些研究，以确定这些和其他关键的 IMC组件能够执行其功能。首先，我们将确定新的顶帽蛋白的作用， 是AC 9/AC 10/ERK 7复合物的假定相互作用物，并评估其在顶帽功能中的作用， 圆锥装配然后，我们将探讨IMC 32如何与新发现的合作伙伴IMC 48合作， 在寄生虫分裂的最早阶段发挥作用，并利用这些蛋白质进一步探索早期的子体 芽蛋白质组最后，我们将利用我们最近开发的光反应非天然氨基酸系统以及 作为冷冻电子断层扫描，以确定寄生虫细胞骨架的关键肺泡是如何组织的， 作为细胞器的支架。总之，这个项目将促进对 弓形虫IMC的结构和功能。由于这种细胞器是寄生虫特异性的， 人类宿主，精确地确定这些重要的IMC组件如何发挥作用， 针对T.弓形虫和其他顶复门寄生虫。