Regulation mechanisms of Trypanosoma brucei axonemal dynein

项目摘要/摘要 运动性对许多寄生虫的生命周期和致病性至关重要。虽然将运动作为目标是成功的 多种细菌性疾病的治疗，真核病原体的运动性和运动性结构 作为处理目标的研究不足和开发不足。动质体虫，这是一种真核寄生虫，能引起 多种被忽视的热带疾病，表现出独特的鞭毛运动。它们的鞭毛以弯曲的波浪拍打着 从鞭毛的顶端传播到鞭毛的底部。这与几乎所有其他真核生物不同，它们从 从底端到顶端。由于动质体鞭毛弯曲波传播方向在一定条件下切换 化学和环境条件，因为动质体鞭毛的运动元素是 与所有其他真核生物几乎相同，轴丝动力蛋白很可能天生具有独特的调节机制， 驱动鞭毛运动的分子马达调节这种末端到根基的运动。检验这一假说需要 动质体动力蛋白调控机制的定量单分子生物物理表征。 这项研究计划的总体目标是使开发新的动叶绿体治疗方法成为可能。 以动质体鞭毛顶端到底端的运动为目标的相关疾病。这个项目的具体目标是 重点是量化布鲁氏锥虫的轴丝动力蛋白调节机制，这将是 用作动体鞭毛的模型。目的包括刻画力量如何调节人的运动性。 内臂轴丝动力蛋白及其相关轻链和微管蛋白的翻译后修饰 调节外臂轴丝动力蛋白。这一跨学科项目将采用分子生物学(CRISPR/CAS9， 克隆、蛋白质标记)、生化(体外重组、ATPase分析)、基因组和蛋白质组(RNA- SEQ、质谱仪)和生物物理(超快双陷光镊子、全内反射荧光 显微镜)实验方法。收集到的数据将被整合并通过制作 轴丝动力蛋白运动机制的定量生物物理模型。预期的结果将是 开发针对寄生虫运动的泛动态体药物的框架。成功完成 该项目最终将使人们更好地了解致病寄生虫的基本机制。 并可能导致治疗非洲昏睡病、恰加斯病和利什曼病的新疗法。

Project Summary/Abstract Motility is critical to the life cycle and pathogenicity of many parasites. While targeting motility is successful in the treatment of multiple bacterial diseases, the motility and motile structures of eukaryotic pathogens remain understudied and underexploited as treatment targets. Kinetoplastids, which are eukaryotic parasites that cause multiple neglected tropical diseases, exhibit unique flagellar motility. Their flagella beat with a bending wave that propagates from the tip to the base of their flagellum. This is unlike nearly all other eukaryotes, which beat from the base to the tip. Because kinetoplastid flagellum bending wave propagation direction switches under certain chemical and environmental conditions, and because the motile elements of kinetoplastid the flagellum are nearly identical to all other eukaryotes, it is likely that unique regulation mechanisms innate to axonemal dyneins, the molecular motors that drive flagellar motility, tune this tip-to-base motility. Testing this hypothesis requires quantitative single-molecule biophysical characterization of kinetoplastid dynein regulation mechanisms. The broad goal of this research program is to enable the development of novel treatments for kinetoplastid- associated diseases that target the tip-to-base motility of kinetoplastid flagella. The specific aims of this project focus on quantifying axonemal dynein regulation mechanisms from Trypanosoma brucei brucei, which will be used as a model for kinetoplastid flagella. The aims include characterizing how force regulates the motility of inner arm axonemal dyneins and how dynein-associated light chains and posttranslational modification to tubulin regulate outer arm axonemal dyneins. This interdisciplinary project will take molecular biological (CRISPR/Cas9, cloning, protein tagging), biochemical (in vitro reconstitutions, ATPase assays), genomic and proteomic (RNA- Seq, mass spec), and biophysical (ultrafast dual-trap optical tweezers, total internal reflectance fluorescence microscopy) experimental approaches. The collected data will be integrated and understood by making quantitative biophysical models of axonemal dynein motility mechanisms. The expected outcome will be a framework from which to develop pan-kinetoplastid drugs that target parasite motility. Successful completion of the project will ultimately lead to a greater understanding of the fundamental mechanisms of pathogenic parasite motility and could lead to novel treatments for African sleeping sickness, Chagas disease, and leishmaniasis.