Chemo-proteomic discovery of lipases in asexual and sexual erythrocytic stages of Plasmodium falciparum

恶性疟原虫无性和有性红细胞阶段脂肪酶的化学蛋白质组学发现

• 批准号: 9374306
• 负责人: Michael Klemba
• 金额: $ 18.71万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-25 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9374306
• 关键词: Affinity; Antimalarials; Area; Artemisinins; Biochemical; Biochemical Genetics; Biochemical Process; Bioinformatics; Biological; Blood; Catabolism; Cell membrane; Cessation of life; Clinical; Combined Modality Therapy; Complex; Culicidae; Development; Disease; Endocytic Vesicle; Enzymes; Epitopes; Erythrocytes; Fatty Acids; Food; Gene Targeting; Generic Drugs; Genes; Hemoglobin; Human; Hydrolase; Hydrolysis; Lead; Life Cycle Stages; Lipase; Lipids; Malaria; Mass Spectrum Analysis; Membrane; Metabolic; Metabolism; Molecular Weight; Parasite resistance; Parasites; Pathway interactions; Phase; Phospholipids; Plasmodium falciparum; Play; Process; Proteome; Proteomics; Public Health; Reaction; Regimen; Resistance; Role; Serine Hydrolase; Source; Transport Vesicles; Triglycerides; Vacuole; asexual; base; drug discovery; genetic approach; inhibitor/antagonist; insight; killings; lipid biosynthesis; novel; novel therapeutics; pathogen; programs; transmission process

ABSTRACT Malaria is a major public health burden in many tropical regions of the world, causing a half million deaths and two hundred million clinical episodes every year. The utility of the first-line anti-malarial regimen, artemisinin combination therapy (ACT), is threatened by emerging resistance. Development of mechanistically distinct anti-malarials will be necessary to replace ACT once parasite resistance inevitably becomes widespread. Understanding fundamental biochemical processes that underpin both asexual and sexual parasite stages in the human host erythrocyte will stimulate the discovery of new drugs to treat disease and block transmission. This proposal seeks to accelerate the discovery and functional characterization of lipases, enzymes that catalyze the hydrolysis of a large, diverse class of biological molecules termed lipids. In Aim 1, a serine hydrolase activity-based probe will be employed to affinity-purify putative parasite lipases, which will then be identified using mass spectrometry. In a complementary approach, two high molecular weight putative lipases will be epitope tagged to assess their contributions to lipolytic transformations in the asexual malaria parasite. To establish the importance of the lipases uncovered in the discovery phase, the genes encoding the enzymes will be targeted for gene disruption. In Aim 2, the discovery of lipases in early and mature gametocytes will be accomplished using the activity-based probe/inhibitor competition approach that has proven successful in asexual parasites. These studies will provide a picture of the diversity of lipolytic serine hydrolases in asexual parasites and in developing and mature gametocytes. They will reveal critical and potentially druggable enzymes and may lead to the discovery of novel biochemical processes that allow the malaria parasite to flourish in human erythrocytes.

摘要 疟疾是世界上许多热带地区的主要公共卫生负担， 每年有2亿例临床病例一线抗疟药的效用 青蒿素联合疗法（ACT）受到新出现的耐药性的威胁。发展 一旦寄生虫产生抗药性， 不可避免地会蔓延开来。了解基础生物化学过程， 人类宿主红细胞中的无性和有性寄生虫阶段将刺激发现 治疗疾病和阻断传播的新药。这项提案旨在加速发现 和脂肪酶的功能特性，酶催化水解的大，不同的， 称为脂质的一类生物分子。在目标1中，将使用基于丝氨酸水解酶活性的探针。 用于亲和纯化推定的寄生虫脂肪酶，然后使用质谱鉴定 光谱法在一种互补的方法中，两种高分子量推定的脂肪酶将被 表位标记，以评估其对无性疟疾中脂解转化的贡献 寄生虫为了确定在发现阶段发现的脂肪酶的重要性， 编码这些酶的基因将成为基因破坏的目标。在目的2中，脂肪酶的发现在早期 和成熟的配子体将完成使用基于活性的探针/抑制剂竞争 这种方法在无性寄生虫中被证明是成功的。这些研究将提供一个图片的 脂肪分解丝氨酸水解酶在无性寄生虫和发育和成熟中的多样性 配子体它们将揭示关键的和潜在的可药物化的酶，并可能导致 发现了新的生物化学过程，使疟疾寄生虫在人体内繁殖 红细胞