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

摘要 疟疾是世界许多热带地区的主要公共卫生负担，造成50万人死亡。 每年死亡和两亿次临床发作。一线抗疟药的效用 青蒿素联合疗法(ACT)正受到新出现的耐药性的威胁。发展 一旦产生寄生虫耐药性，将需要使用机械上不同的抗疟疾药物来取代ACT 不可避免地会变得普遍。了解构成基础的基本生化过程 人类宿主红细胞中的无性和有性寄生虫阶段都将刺激发现 治疗疾病和阻断传播的新药。这项提议旨在加速这一发现 以及脂肪酶的功能特性，脂肪酶催化大量、多样化的 一类被称为脂类的生物分子。在目标1中，一个基于丝氨酸水解酶活性的探针将 被用来亲和纯化假定的寄生虫脂肪酶，然后使用质谱仪进行鉴定 光谱分析。在一种互补的方法中，两种可能的高分子量脂肪酶将是 标记表位以评估它们在无性疟疾中对脂解转化的贡献 寄生虫。为了确定在发现阶段发现的脂肪酶的重要性，这些基因 编码这些酶将成为基因破坏的目标。在目标2中，脂肪酶在早期的发现 成熟的配子体将通过基于活性的探针/抑制物竞争来完成 已被证明在无性寄生虫身上取得成功的方法。这些研究将提供一幅关于 无性寄生虫和发育成熟期脂解丝氨酸水解酶的多样性 配子体。它们将揭示关键的和潜在的可下药的酶，并可能导致 发现新的生化过程使疟疾寄生虫在人类体内繁衍 红血球。

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.