Structure-function relationships of kinesin molecular motors in Plasmodium parasites

运动蛋白是基于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。可获得的药物很少，而且出现耐药性，这也意味着迫切需要新的药物靶点。有丝分裂运动蛋白是癌症治疗的靶点，也可能成为抗疟疾药物的有吸引力的靶点。我们对疟原虫的运动蛋白知之甚少，因此本项目的目的是结合结构、生化和寄生虫学方法来对这些马达进行机械和功能表征。本项目将研究疟原虫运动蛋白的结构-功能关系，重点研究运动蛋白13和运动蛋白8。他们的分子机制，生物学作用，和敏感性的抑制，主要是在恶性疟原虫，将进行调查。包括疟原虫在内的所有真核生物的微管都经历了剧烈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13s和驱动蛋白8s——与微管重塑有关，反映了典型驱动蛋白步进机制的一个重要但鲜为人知的差异。已知动力学蛋白13s是微管解聚合剂，利用ATP缩短微管；我们已经证明，恶性疟原虫和人类的酪蛋白-13马达的重组亚域都能解聚微管。然而，它们活动的细节表明，每种生物的马达在机制上存在差异。动力蛋白-8的机制更有争议。因此，有必要发现疟原虫与高等生物的激酶之间的序列同源性/差异性是如何在机制上反映出来的。差异可能使寄生虫酶容易受到抗疟疾治疗的特异性抑制。我们将使用低温电子显微镜对运动机制进行结构表征。另一个核心问题是单个动力蛋白在寄生虫中扮演什么角色。包括锌指核酸酶技术在内的用于控制恶性疟原虫遗传上具有挑战性的新技术将使人们能够有效地评估动力蛋白在疟原虫中的作用。该项目的目标是：1)纯化重组恶性疟原虫激酶-13和激酶-8马达结构域；2)利用低温电子显微镜对它们及其微管相互作用进行生化/结构表征；3)利用小分子文库筛选抑制剂；4)确定kinesin-13/8的表达谱，了解其生物学作用；5)比较不同疟原虫种间kinesin-13/8序列；6)在恶性疟原虫中产生修饰的kinesin-13/8转基因寄生虫系，明确其功能。因此，本项目将涉及细胞内寄生虫的分子机制和功能贡献的研究。

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.