MITOPlas_Scalable characterization of the malaria parasite mitochondrial proteome

 描述(由申请人提供)：我们有效治疗疟疾的能力受到对现有数量有限的一线抗疟疾药物日益广泛的耐药性的威胁。因此，迫切需要新的战略来指导药物开发新目标的优先次序。在这里，我们建议将线粒体功能作为一种战略，以产生治疗疟疾的不同解决方案。长期目标是确定人类疟疾寄生虫恶性疟原虫的线粒体蛋白质组，并建立这些蛋白质的核心子集，这些蛋白质是寄生虫生存所必需的。我们设想，这些知识将有助于开发具有新作用机制的新的抗疟疾药物，并绕过现有的耐药模式。本研究的目标是：(1)开发一条管道，用于优先排序并验证被交易到线粒体的一组核编码蛋白质；以及(2)对这些蛋白质的重要性进行分类。核心假设是线粒体功能对疟疾寄生虫在血液、蚊子和肝脏阶段的生存至关重要。定义必要的线粒体蛋白质和生物过程应该会为基础寄生虫生物学提供新的见解。此外，这可以为开发同时针对寄生虫生命周期的血液、传播和肝脏阶段的抗疟疾药物创造机会。具有这些特征的药物对消除疟疾的努力至关重要。为了实现这项建议的目标，我们将追求两个目标。在具体目标1中，我们将使用生物信息学工具来预测核编码蛋白质中的线粒体靶向序列(MTS)，并创建假定的线粒体蛋白质的优先列表。我们将创建与荧光报告蛋白融合的候选MTS文库，并使用高通量和高含量成像来确定候选MTS是否足以介导蛋白质运输到线粒体。在具体目标2中， 我们将使用一种新开发的功能遗传学工具包来表位、标记和有条件地调节假定运输到线粒体的内源性蛋白的表达水平。这将使我们能够确定与给定的MTS相关的天然蛋白质是否确实被贩运到线粒体。有条件地调节这些蛋白质的表达还将有助于评估哪些线粒体蛋白质是寄生虫生存所必需的。我们的方法是创新的，因为它集成了几种互补的技术-组合报告文库合成和寄生虫系生成、高内容成像和功能遗传学-以前所未有的规模深入了解恶性疟原虫的线粒体生物学。这项拟议的研究意义重大，因为它将为这种相对神秘的寄生虫细胞器的组成和重要成分提供明确的见解。同时，它将激发对线粒体功能的有针对性的干扰，作为识别生命周期阶段非依赖性抗疟疾药物的一种战略。预计这些成果将通过改进现有的疟疾治疗和根除工具，对人类健康产生积极影响。

 DESCRIPTION (provided by applicant): Our ability to effectively treat malaria is threatened by increasingly widespread resistance to the limited number of frontline antimalarial drugs available. Consequently, new strategies guiding prioritization of novel targets for drug development are critically needed. Here we propose targeting mitochondrial function as a strategy that could yield diverse solutions for treating malaria. The long-term goal is to define the mitochondrial proteome of the human malaria parasite, P. falciparum, and establish the core subset of these proteins that are essential for parasite survival. We envision that this knowledge will contribute to the development of new antimalarial drugs with novel mechanisms of action, and that circumvent existing modes of drug resistance. The objectives of the present research are to: (1) develop a pipeline for prioritizing then validating the set of nuclear- encoded protein that are trafficked to the mitochondrion; and (2) classify the essentiality of these proteins. The central hypothesis is that mitochondrial function is critical for malaria parasite survival during blood, mosquito and liver stages. Defining essential mitochondrial proteins and biological processes should provide new insights into fundamental parasite biology. Additionally, this can create opportunities for developing antimalarial drugs that simultaneously target blood, transmission, and liver stages of the parasite life cycle. Drugs with these characteristics are critical to malaria elimination efforts. To accomplish the objectives of this proposal, we will pursue two aims. In Specific Aim 1, we will use bioinformatics tools to predict mitochondrial targeting sequences (MTS) in nuclear-encoded proteins, and create prioritized lists of putative mitochondrial proteins. We will create libraries of MTS candidates fused to a fluorescent reporter protein and use high throughput and high content imaging to determine whether a candidate MTS is sufficient to mediate protein trafficking to the mitochondrion. In Specific Aim 2, we will use a newly developed functional genetics toolkit to epitope tag and conditionally regulate the expression level of endogenous proteins putatively trafficked to the mitochondrion. This will allow us to establish whether native proteins associated with a given MTS are indeed trafficked to the mitochondrion. Conditionally regulating the expression of these proteins will additionally facilitate assessment of which mitochondrial proteins are essential for parasite survival. Our approach is innovative because it integrates several complementary technologies-combinatorial reporter library synthesis and parasite line generation, high content imaging and functional genetics-to gain insight into mitochondrial biology on an unprecedented scale in P. falciparum. The proposed research is significant because it will provide definitive insights into the composition and vital components of this relatively enigmatic parasite organelle. Simultaneously, it will motivate targeted interference with mitochondrial function as a strategy for identifying life cycle stage-independent antimalarial drugs. These outcomes are expected to positively impact human health by improving the tools available for malaria treatment and eradication.