MOLECULAR MECHANISM OF THE CYTOPLASMIC DYNEIN-DYNACTIN MOTOR COMPLEX

细胞质动力蛋白-动力蛋白运动复合物的分子机制

基本信息

批准号：
8892200
负责人：
Arne Gennerich
金额：
$ 2.64万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-01 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8892200
关键词：
ATP phosphohydrolase ATPase Domain Affect Affinity Behavior Binding Biological Assay Cell physiology Complex Cytoskeleton Disease Drosophila genus Dynein ATPase Energy Transfer Etiology Eukaryotic Cell Fluorescence Fluorescence Microscopy Foundations Functional disorder Future Generations Genetic Glycine Goals Grant Health Human Hydrolysis Kinesin Knowledge Length Link Measurement Measures Mechanics Methods Microtubules Minus End of the Microtubule Molecular Motion Motor Multiprotein Complexes Mutagenesis Mutation Myosin ATPase Nucleotides Physiology Positioning Attribute Production Property Protein Engineering Protein Family Proteins Recombinants Role Saccharomyces cerevisiae Shapes Site Slide Source Structure Syndrome Testing Therapeutic Therapeutic Intervention Time Walking Weight-Bearing state Work Yeasts base biochemical tools cofactor crosslink dimer dynactin experience genetic regulatory protein human disease insight laser tweezer motor neuron degeneration mutant nervous system disorder optical traps particle response single molecule stem tool

项目摘要

DESCRIPTION (provided by applicant): Our long-term goal is to elucidate the molecular mechanism of the cytoplasmic dynein-dynactin motor complex, and to define the molecular bases of dynein-related diseases in humans. Dynein is the primary vehicle for microtubule minus-end-directed transport in eukaryotic cells. The function and dysfunction of this vital motor and its regulatory proteins contribute to a broad set of cellular functions and human diseases. Despite increasing efforts to define dynein's functional properties, the molecular mechanisms that govern dynein's mechanochemistry remain poorly understood. This deficiency largely stems from dynein's structural complexity. Dynein belongs to the AAA+ class of ATP-hydrolyzing mechanoenzymes that assemble into ring- shaped structures, and therefore, possesses characteristically distinct structural features compared to the other two cytoskeletal motor protein families, kinesin and myosin. Dynein is also exceptionally large (~1.2 MDa) and structure-function studies on dynein have been limited by the availability of functional recombinant dynein. Adding to dynein's complexity, dynein associates with multiple accessory chains and the dynactin complex, all of which are essential for nearly every cellular function of dynein. Mutations in dynactin's largest subunit, p150glued, which contains dynactin's putative microtubule-binding domain, cause Perry syndrome and motor neuron degeneration in humans. Yet, the role of p150glued in dynein function remains unknown. In this grant, we seek to overcome these limitations by combining ultrasensitive single-molecule assays with protein engineering. We will use S. cerevisiae, the only source for recombinant full-length dynein and dynactin, to produce stable wildtype and mutant versions of both multiprotein complexes. Using these biochemical tools and multicolor single-molecule fluorescence and optical tweezers methods, we will resolve 1) how dynein's AAA+ motor domains are coordinated within dynein's mechanochemical cycle, 2) how dynactin modulates and regulates dynein function, and 3) how human p150glued mutations disrupt the function of the dynein-dynactin complex. This information will provide insight into cellular physiology and pathophysiology, and potentially identify targets within the dynein-dynactin complex for therapeutic interventions.

描述（由申请人提供）：我们的长期目标是阐明细胞质动力蛋白-动力蛋白运动复合物的分子机制，并定义人类动力蛋白相关疾病的分子基础。动力蛋白是真核细胞中微管负端定向运输的主要载体。这种重要运动及其调节蛋白的功能和功能障碍导致了广泛的细胞功能和人类疾病。尽管人们越来越努力地定义动力蛋白的功能特性，但控制动力蛋白机械化学的分子机制仍然知之甚少。这种缺陷很大程度上源于动力蛋白的结构复杂性。动力蛋白属于 AAA+ 类 ATP 水解机械酶，可组装成环形结构，因此与其他两个细胞骨架运动蛋白家族（驱动蛋白和肌球蛋白）相比，具有独特的结构特征。动力蛋白也非常大（~1.2 MDa），并且动力蛋白的结构功能研究受到功能性重组动力蛋白可用性的限制。动力蛋白与多个辅助链和动力蛋白复合物结合，增加了动力蛋白的复杂性，所有这些对于动力蛋白的几乎所有细胞功能都是必需的。 dynactin 最大的亚基 p150glued（包含 dynactin 推定的微管结合域）的突变会导致人类佩里综合征和运动神经元变性。然而，p150glued 在动力蛋白功能中的作用仍然未知。在这笔资助中，我们寻求通过将超灵敏单分子测定与蛋白质工程相结合来克服这些限制。我们将使用重组全长动力蛋白和动力蛋白的唯一来源酿酒酵母来生产两种多蛋白复合物的稳定野生型和突变体版本。使用这些生化工具以及多色单分子荧光和光镊方法，我们将解决 1) 动力蛋白的 AAA+ 运动域如何在动力蛋白的机械化学循环中协调，2) 动力蛋白如何调节和调节动力蛋白功能，以及 3) 人类 p150 粘合突变如何破坏动力蛋白-动力蛋白复合物的功能。这些信息将提供对细胞生理学和病理生理学的深入了解，并有可能确定动力蛋白-动力蛋白复合物内的靶点以进行治疗干预。