Structure-function relationships of kinesin molecular motors in Plasmodium parasites

战略研究重点：世界一流的生物科学摘要驱动蛋白是一种依赖ATP的微管马达，在真核生物包括寄生原生动物中具有多种重要作用。细胞内疟原虫每年感染超过2亿人，造成近100万人死亡。复杂的寄生虫生命周期对细胞生物学的教科书概念提出了许多挑战。可用的药物很少和新出现的耐药性也意味着迫切需要新的药物靶点。有丝分裂驱动蛋白是癌症治疗的靶点，并可能成为抗疟疾药物的有吸引力的靶点。疟原虫中的驱动蛋白知之甚少，因此本项目的目的是将联合收割机结构、生物化学和寄生虫学方法结合起来，从机械和功能上表征这些电机。本项目将阐明疟原虫驱动蛋白的结构与功能关系，重点是驱动蛋白-13和驱动蛋白-8马达。它们的分子机制，生物学作用，和抑制的敏感性，主要是在恶性疟原虫，将进行研究。 包括疟原虫在内的所有真核生物中的微管都经历了戏剧性的细胞周期依赖性重组，特别是在细胞分裂期间。驱动蛋白的一个子集-主要是驱动蛋白-13和驱动蛋白-8-参与微管重塑，反映了典型驱动蛋白步进机制的重要但知之甚少的分歧。已知驱动蛋白-13是微管解聚剂，使用ATP缩短微管;我们已经表明，来自恶性疟原虫和人类的驱动蛋白-13马达的重组子结构域解聚微管。然而，它们的活动细节表明，每种生物体的马达之间存在机械差异。驱动蛋白-8的机制更具争议性。 这是至关重要的，以发现疟原虫驱动蛋白和那些从高等生物之间的序列同源性/分歧是如何反映机制。差异可能使寄生虫酶易受抗疟疾治疗的特异性抑制。我们将使用低温电子显微镜来表征电机机制的结构。另一个中心问题是驱动蛋白在寄生虫中扮演什么角色。用于操纵具有遗传挑战性的恶性疟原虫的新技术-包括锌指核酸酶技术-将能够有效评估驱动蛋白在疟原虫中的作用。 本项目的主要目的是：1）纯化重组恶性疟原虫驱动蛋白-13和驱动蛋白-8的马达结构域; 2）利用冷冻电镜对其进行生物化学/结构表征及其微管相互作用; 3）利用小分子文库筛选抑制剂; 4）确定驱动蛋白-13/8的表达谱以了解其生物学作用; 6）在恶性疟原虫中产生经修饰的驱动蛋白-13/8的转基因寄生虫系以指定其功能。因此，本计画将探讨驱动蛋白在胞内寄生虫中的分子机制与功能贡献。

Strategic Research Priority: World Class BioscienceAbstract Kinesins are ATP-dependent microtubule-based motors with multiple essential roles in eukaryotes, including parasitic protozoa. The intracellular Plasmodium parasite infects >200 million people annually, killing nearly 1 million. The complex parasite life cycle presents numerous challenges to text-book concepts of cell biology. The few drugs available and emerging drug resistance also mean that novel drug targets are urgently needed. Mitotic kinesins are targets for cancer therapeutics and could form attractive targets for anti-malarials. Little is known about kinesins in Plasmodium, so the aim of this project is to combine structural, biochemical and parasitology approaches to mechanistically and functionally characterize these motors. Project This project will elucidate the structure-function relationship of Plasmodium kinesins, with a focus on kinesin-13 and kinesin-8 motors. Their molecular mechanisms, biological roles, and sensitivity to inhibition, primarily in P. falciparum, will be investigated. Microtubules in all eukaryotes, including Plasmodium, undergo dramatic cell cycle-dependent reorganisation, particularly during cell division. A subset of kinesins - principally kinesin-13s and kinesin-8s - are implicated in microtubule remodeling and reflect an important, but poorly understood, divergence of the typical kinesin stepping mechanism. Kinesin-13s are known to be microtubule depolymerisers, using ATP to shorten microtubules; we have shown that recombinant subdomains of kinesin-13 motors from both P. falciparum and humans depolymerise microtubules. However, the specifics of their activities suggest there are mechanistic differences between motors from each organism. The kinesin-8 mechanism is more controversial. It is essential to discover how sequence homology/divergence between Plasmodium kinesins and those from higher organisms is reflected mechanistically. Differences potentially render parasite enzymes susceptible to specific inhibition by anti-malaria treatments. We will use cryo-electron microscopy to structurally characterize motor mechanisms. Another central question is what role individual kinesins play in the parasite. Novel technologies for manipulating the otherwise genetically challenging P. falciparum - including Zinc Finger Nuclease Technology - will enable efficient assessment of the role of kinesins in Plasmodium. The objectives of the project are: 1) Purify recombinant P. falciparum kinesin-13 and kinesin-8 motor domains; 2) Biochemically/structurally characterize them and their microtubule interaction using cryo-electron microscopy; 3) Screen for inhibitors using small molecule libraries; 4) Determine expression profiles of kinesin-13/8 to understand their biological roles; 5) Compare sequences of kinesin-13/8 across Plasmodium species; 6) Generate transgenic parasite lines of modified kinesin-13/8 in P. falciparum to specify their functions. Thus, this project will involve the study of both the molecular mechanisms and functional contribution of kinesins in intracellular parasites.