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Molecular mechanisms of sporogonic development in malaria parasites

疟原虫孢子发育的分子机制

基本信息

批准号：
MR/P021611/1
负责人：
Johannes Dessens
金额：
$ 58.8万
依托单位：
London School of Hygiene & Tropical Medicine
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2018
资助国家：
英国
起止时间：
2018 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=MR%2FP021611%2F1
关键词：
Molecular mechanisms sporogonic development malaria

项目摘要

New therapies for prevention and treatment of malaria, and for reducing transmission, are urgently needed. This project aims to increase our understanding of the molecular mechanisms underlying the formation and function of the crystalloid - a malaria parasite organelle found uniquely in the ookinete and young oocyst stages that is essential for sporogonic development and sporozoite transmission from mosquito to human. A palmitoyl-S-acyl transferase enzyme named DHHC10 resides in the crystalloid and is essential for crystalloid formation and parasite transmission. DHHC10 catalyses the addition of a palmitoyl lipid to proteins, a reaction known as palmitoylation. This project will focus on the role of palmitoylation in crystalloid biogenesis and function. Using the Plasmodium berghei mouse malaria model, we will determine the substrate range, spatiotemporal dynamics and mechanisms of DHHC10-mediated palmitoylation in crystalloids via three specific objectives: Objective 1: Determine the crystalloid palmitome. Using a method called acyl-biotin-exchange (ABE) the palmitoyls on palmitoylated proteins are chemically exchanged for biotin, which allows their specific capture and purification with microbeads that bind biotin. Following this, the palmitoylated protein fraction (palmitome) is analyzed by mass spectrometry (MS) to determine the identities of the individual protein components. By comparing the palmitomes from ookinetes that express DHHC10 and those that do not (DHHC10-knockouts), the substrates of DHHC10 can be identified, which correspond to the crystalloid palmitome. In parallel, the ookinete palmitome of a DHHC-positive, but crystalloid-negative, parasite line (LAP3-knockout) will be determined to study the spatiotemporal dynamics of DHHC10-mediated palmitoylation. The results will provide a global view of the palmitoylated protein repertoire in the whole ookinete and in the crystalloid, and will identify new crystalloid components.Objective 2: Identify proteins that interact with DHHC10. Proteins that interact with DHHC10 (either subunits of a DHHC10 protein complex, or DHHC10 substrates) will be identified by two approaches. (i) GFP pull-down: ookinetes that express DHHC10 fused to green fluorescent protein (GFP) will be used to capture DHHC10 protein complexes using magnetic beads that can bind to the GFP, and these complexes will then be analyzed by MS to determine their protein composition. (ii) BioID: ookinetes that express DHHC10 fused to a biotin ligase (BirA*) can be used to specifically biotinylate DHHC10 and its neighbour proteins upon addition of excess biotin to the cells. This allows their specific capture with magnetic beads that bind to biotin, followed by MS analysis. BioID analysis of ookinetes expressing a different crystalloid protein (LAP3) fused to BirA* will be carried out to validate the DHHC10 results. The results will identify new protein partners and substrates of DHHC10, as well as new crystalloid components, and provide new insight into the spatiotemporal dynamics and mechanisms of DHHC10-mediated palmitoylation. Objective 3: Validate new candidate crystalloid proteins. To make sure that newly identified proteins of the crystalloid palmitome and DHHC10 interactome are genuinely associated with crystalloid biogenesis and/or function, they will be functionally characterized using fluorescent protein tagging and gene knockout approaches in genetically modified parasites. The combined results obtained from this project will build a better and more comprehensive picture of the molecules that are associated with the crystalloid organelle and are involved in, or subject of, crystalloid-specific S-palmitoylation. They will provide important new insight into the molecular mechanisms that facilitate crystalloid genesis and function, and form a platform for the identification of new drug targets specific for sporogonic development, providing new strategies to prevent transmission.

迫切需要预防和治疗疟疾以及减少传播的新疗法。这个项目旨在增加我们对晶体形成和功能的分子机制的理解--晶体是一种疟疾寄生虫细胞器，独特地存在于卵动细胞和幼年卵囊阶段，对于孢子体发育和子孢子从蚊子传播到人类是必不可少的。棕榈酰-S-酰基转移酶DHHC10存在于晶体中，对晶体的形成和寄生虫的传播是必不可少的。DHHC10催化蛋白质加成棕榈酰基脂，这一反应称为棕榈酰化。本项目将侧重于棕榈酰化在晶体生物发生和功能中的作用。利用伯氏疟原虫小鼠疟疾模型，我们将通过三个特定的目标来确定DHHC10介导的棕榈酰化的底物范围、时空动力学和机制：目的1：确定晶体棕榈酰化。使用一种名为酰基-生物素交换(ABE)的方法，棕榈酰化蛋白质上的棕榈酰基被化学交换为生物素，这使得它们能够通过结合生物素的微珠进行特定的捕获和纯化。然后，用质谱仪(MS)分析棕榈酰化蛋白质组分(Palmitome)，以确定单个蛋白质组分的身份。通过比较表达DHHC10和不表达DHHC10的动子的棕榈体，可以确定DHHC10的底物，它们对应于晶体棕榈体。同时，将通过测定DHHC阳性但晶体阴性的寄生虫系(LAP3-基因敲除)的动细胞掌状体来研究DHHC10介导的棕榈酰化的时空动力学。结果将提供整个动子和晶体中棕榈酰化蛋白质谱的全局视图，并将识别新的晶体成分。目标2：鉴定与DHHC10相互作用的蛋白质。与DHHC10(DHHC10蛋白复合体的亚基或DHHC10底物)相互作用的蛋白质将通过两种方法进行鉴定。(I)GFP下拉：表达DHHC10与绿色荧光蛋白(GFP)融合的动子将被用来通过可与GFP结合的磁珠捕获DHHC10蛋白质复合体，然后通过MS分析这些复合体以确定其蛋白质组成。(Ii)BioID：表达DHHC10与生物素连接酶(BIRA*)融合的动子可用于在细胞中加入多余的生物素时，特异性地生物素化DHHC10及其邻近蛋白质。这使得它们可以通过与生物素结合的磁珠进行特定捕获，然后进行MS分析。将对融合到BIRA*的表达不同晶体蛋白(LAP3)的动子进行生物ID分析，以验证DHHC10的结果。这些结果将确定DHHC10的新的蛋白质配对和底物，以及新的晶体成分，并为研究DHHC10介导的棕榈酰化的时空动力学和机制提供新的见解。目的3：验证新的候选晶体蛋白。为了确保新发现的晶体棕榈体和DHHC10相互作用体的蛋白质真正与晶体生物发生和/或功能相关，将在转基因寄生虫中使用荧光蛋白标签和基因敲除方法对它们进行功能表征。这一项目的综合结果将更好、更全面地描绘与晶体细胞器相关的、参与或参与晶体特异性S棕榈酰化的分子。它们将为促进晶体发生和功能的分子机制提供重要的新见解，并形成一个平台，用于识别针对造孢子体发育的新药物靶点，提供防止传播的新策略。