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 - 二奈氏菌运动复合物的分子机制，并定义人类中动蛋白相关疾病的分子碱基。动力蛋白是真核细胞中微管减去指导转运的主要载体。该重要运动及其调节蛋白的功能和功能障碍有助于广泛的细胞功能和人类疾病。尽管越来越多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很少了解。这种缺乏很大程度上源于动力蛋白的结构复杂性。 Dynein属于AAA+类的ATP - 氢化酶酶，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，它们组装成环形结构。动力蛋白也非常大（〜1.2 MDA），并且对动力蛋白的结构功能研究受到功能重组动力蛋白的可用性受到限制。除了Dynein的复杂性外，Dynein与多个附属链和Dynactin复合物相关联，所有这些都对Dynein的几乎每个细胞功能都是必不可少的。 Dynactin最大的亚基中的突变P150Glued包含Dynactin的假定微管结合结构域，引起了人类的佩里综合征和运动神经元变性。然而，p150gly在动力蛋白功能中的作用仍然未知。在这笔赠款中，我们试图通过将超敏化单分子测定与蛋白质工程相结合来克服这些局限性。我们将使用S. cerevisiae，这是重组全长动力蛋白和dynactin的唯一来源，以产生两种多蛋白复合物的稳定野生型和突变版本。 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.该信息将提供对细胞生理学和病理生理学的见解，并有潜在地识别动力蛋白 - 二奈氏蛋白复合物中的靶标的治疗干预措施。