MITOPlas_Scalable characterization of the malaria parasite mitochondrial proteome

MITOPlas_疟原虫线粒体蛋白质组的可扩展表征

• 批准号: 9014806
• 负责人: JACQUIN C NILES
• 金额: $ 23.4万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9014806
• 关键词: Acute; Antimalarials; Bioinformatics; Biological Assay; Biological Process; Biology; Blood; CRISPR/Cas technology; Categories; Cessation of life; Characteristics; Communities; Culicidae; Development; Disease; Drug Targeting; Drug resistance; Due Process; Elements; Epitopes; Functional Imaging; Gene Expression; Generations; Genes; Genetic; Genome engineering; Goals; Health; Human; Image; Immunofluorescence Immunologic; Impairment; Knowledge; Lead; Libraries; Life Cycle Stages; Liver; Maintenance; Malaria; Mediating; Mission; Mitochondria; Mitochondrial Proteins; Morbidity - disease rate; Nuclear; Organelles; Outcome; Parasites; Pharmaceutical Preparations; Plasmodium; Plasmodium falciparum; Production; Protein Import; Proteins; Proteome; Public Health; Regimen; Reporter; Research; Resistance; Resources; Set protein; Staging; Technology; Therapeutic; Time; Translating; United States National Institutes of Health; Vaccines; burden of illness; combinatorial; drug development; drug discovery; fitness; fluorescence imaging; improved; innovation; insight; mitochondrial metabolism; mortality; novel; novel therapeutics; pathogen; prevent; protein expression; protein transport; symptom treatment; therapeutic development; tool; trafficking; transmission process

 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.

 描述（由适用提供）：我们有效治疗疟疾的能力受到对有限数量的前线抗疟药的广泛抵抗的威胁。因此，至关重要的是指导新型药物开发目标的新策略。在这里，我们提出靶向线粒体功能作为一种可以产生疟疾解决方案的策略。长期的目标是定义人类疟原虫，恶性疟原虫的线粒体蛋白质组，并确定这些蛋白质的核心子集，这对于寄生虫生存至关重要。我们设想，这种知识将有助于采用新型的作用机理发展新的抗疟药，并规避现有的耐药性模式。本研究的目标是：（1）开发一条管道，以确定优先级，然后验证被贩运到线粒体的核编码蛋白的集合； （2）分类这些蛋白质的本质。中心假设是线粒体功能对于血液，蚊子和肝脏阶段期间疟疾寄生虫存活至关重要。定义基本的线粒体蛋白和生物过程应提供有关基本寄生虫生物学的新见解。此外，这可以为开发仅针对寄生虫生命周期的血液，传播和肝脏阶段的抗疟药创造机会。具有这些特征的药物对于消除疟疾的工作至关重要。为了实现该提议的目标，我们将追求两个目标。在特定目标1中，我们将使用生物信息学工具来预测核编码蛋白中的线粒体靶向序列（MTS），并创建推定的线粒体蛋白的优先列表。我们将创建与荧光报告蛋白融合的MTS候选者文库，并使用高吞吐量和高内容成像来确定候选MTS是否足以将蛋白质运输介导到线粒体上。在特定的目标2中， 我们将使用新开发的功能遗传学工具包来表位标签，并​​有条件调节被交通于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MT相关的天然蛋白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将进一步支持评估哪些线粒体蛋白对于寄生虫生存至关重要。我们的方法具有创新性，因为它将几种互补的技术与组合记者库的合成和寄生虫线的产生，高含量成像和功能遗传学结合在一起，从而在P. falciparum的前所未有的规模上洞悉了对线粒体生物学的见解。拟议的研究之所以重要，是因为它将提供对这种相对神秘的寄生虫细胞器的组成和重要组成部分的明确见解。同时，它将激励针对性的干扰线粒体功能，以此作为识别生命周期阶段独立的抗疟药的策略。这些结果有望通过改善可用于疟疾治疗和消除的工具对人类健康产生积极影响。