Regulation mechanisms of Trypanosoma brucei axonemal dynein

布氏锥虫轴丝动力蛋白的调控机制

• 批准号: 10494466
• 负责人: Joshua Alper
• 金额: $ 26.12万
• 依托单位: CLEMSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10494466
• 关键词: ATP phosphohydrolase; Affect; African Trypanosomiasis; Bacterial Infections; Behavior; Biochemical; Biochemistry; Biological; Biological Assay; Biophysics; CRISPR/Cas technology; Cell division; Cells; Centers of Research Excellence; Chagas Disease; Chemicals; Chronic; Cilia; Cloning; Data; Development; Disease; Drug Targeting; Drug resistance; Dynein ATPase; Elements; Enzymes; Eukaryota; Exhibits; Flagella; Fluorescence Microscopy; Frequencies; Genomics; Goals; In Vitro; Lead; Leishmaniasis; Life Cycle Stages; Light; Malignant Neoplasms; Measures; Microtubules; Modeling; Modification; Molecular; Molecular Motors; Morphogenesis; Motor; Movement; Outcome; Parasites; Pathogenicity; Persons; Phenotype; Post-Translational Protein Processing; Process; Property; Proteins; Proteomics; Regulation; Research; Structure; Testing; Tissues; Total Internal Reflection Fluorescent; Trypanosoma; Trypanosoma brucei brucei; Trypanosomiasis; Tubulin; Virulence; arm; base; biophysical model; biophysical properties; cell motility; force feedback; innovation; insight; laser tweezer; mechanical signal; neglected tropical diseases; novel; optical traps; pathogen; programs; reconstitution; single molecule; therapeutic target; transcriptome sequencing; transmission process; vector

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.

项目概要/摘要 运动性对于许多寄生虫的生命周期和致病性至关重要。虽然靶向运动是成功的 治疗多种细菌性疾病，保留真核病原体的运动性和运动结构 作为治疗目标尚未得到充分研究和利用。动质体，是一种真核寄生虫，可引起 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的形式跳动 从鞭毛的尖端传播到鞭毛的基部。这与几乎所有其他真核生物不同，它们从 基部到尖端。由于动质体鞭毛弯曲波传播方向在一定条件下发生切换 化学和环境条件，并且由于动质体鞭毛的运动元素是 与所有其他真核生物几乎相同，轴丝动力蛋白可能具有固有的独特调节机制， 驱动鞭毛运动的分子马达，调节这种尖端到基底的运动。检验这个假设需要 动质体动力蛋白调节机制的定量单分子生物物理表征。 该研究计划的总体目标是开发动质体的新疗法 以动质体鞭毛的尖端到基部运动为目标的相关疾病。该项目的具体目标 重点是量化布氏锥虫的轴丝动力蛋白调节机制，这将是 用作动质体鞭毛的模型。目标包括描述力如何调节物体的运动性 内臂轴丝动力蛋白以及动力蛋白相关轻链和微管蛋白的翻译后修饰如何 调节外臂轴丝动力蛋白。这个跨学科项目将采用分子生物学（CRISPR/Cas9、 克隆、蛋白质标签）、生物化学（体外重组、ATP 酶测定）、基因组和蛋白质组学（RNA- Seq、质谱）和生物物理（超快双阱光镊、全内反射荧光 显微镜）实验方法。收集到的数据将通过以下方式进行整合和理解： 轴丝动力蛋白运动机制的定量生物物理模型。预期结果将是 开发针对寄生虫运动的泛动质体药物的框架。顺利完成 该项目最终将导致人们对致病寄生虫的基本机制有更深入的了解 运动性，并可能导致非洲昏睡病、恰加斯病和利什曼病的新疗法。