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.

项目总结/摘要 运动性对许多寄生虫的生命周期和致病性至关重要。虽然靶向运动是成功的 在多种细菌性疾病的治疗中，真核病原体的运动性和运动结构仍然存在， 作为治疗目标的研究和开发不足。动质体是一种真核寄生虫 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跳动， 从鞭毛的顶端向基部传播。这与几乎所有其他真核生物不同， 从底部到尖端由于动体鞭毛弯曲波的传播方向在一定的条件下发生转换， 化学和环境条件，并且由于动质体的运动元件，鞭毛 与所有其他真核生物几乎相同，轴丝动力蛋白先天的独特调节机制， 驱动鞭毛运动的分子马达，调节这种尖端到基部的运动。测试这个假设需要 动质体动力蛋白调节机制的定量单分子生物物理表征。 这项研究计划的主要目标是开发新的动质体治疗方法， 相关疾病的目标运动体鞭毛的尖端到基部的运动。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。目的包括描述力如何调节 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 调节外臂轴丝动力蛋白。这个跨学科项目将采取分子生物学（CRISPR/Cas9， 克隆、蛋白质标记）、生物化学（体外重组、ATP酶测定）、基因组学和蛋白质组学（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。预期的结果将是 框架，从中开发泛动质体药物，目标寄生虫运动。成功完成 该项目将最终导致对致病寄生虫的基本机制有更深入的了解 这可能会导致非洲昏睡病，恰加斯病和利什曼病的新疗法。