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.

项目摘要/摘要 运动对于许多寄生虫的生命周期和致病性至关重要。而定位运动成功 多种细菌疾病的治疗，真核病原体的运动性和运动结构仍然存在 被研究和不流失为治疗靶标。动力质体，是导致的真核生物寄生虫 多种被忽视的热带疾病表现出独特的鞭毛运动。他们的鞭毛用弯曲的波击败 从尖端到鞭毛的底部传播。这与几乎所有其他真核生物都不一样 尖端的基础。因为动力质体鞭毛弯曲波传播方向在某些 化学和环境条件，并且由于鞭毛的动力学元素是 与所有其他真核生物几乎相同，可能是独特的调节机制与轴突动力蛋白的先天机制， 驱动鞭毛运动的分子电动机，调整此尖端到基础运动。检验此假设需要 动力质体动力蛋白调节机制的定量单分子生物物理表征。 该研究计划的广泛目的是使动作塑料的新型治疗方法开发 靶向动作塑料鞭毛的尖端到基础运动的相关疾病。该项目的具体目的 专注于量化Brucei Brucei锥虫的轴突动力蛋白调节机制，这将是 用作动力质体鞭毛的模型。目的包括表征力如何调节运动能力 内臂轴突动力蛋白以及针对微管蛋白的脱氧蛋白相关的光链和翻译后如何修饰 调节外臂轴突动力蛋白。这个跨学科项目将采用分子生物学（CRISPR/CAS9， 克隆，蛋白质标记），生化（体外重构，ATPase分析），基因组和蛋白质组学（RNA- SEQ，质量规格）和生物物理（超快双陷阱光学镊子，总内部反射率荧光 显微镜）实验方法。收集的数据将通过制作来集成和理解 轴突动力蛋白运动机制的定量生物物理模型。预期的结果将是 从中开发出靶向寄生虫运动的泛体形细胞药物的框架。成功完成 该项目最终将对致病寄生虫的基本机制有更深入的了解 运动能力，可能导致对非洲熟睡，查加斯病和利什曼病的新型治疗。