MOLECULAR MECHANISM OF THE CYTOPLASMIC DYNEIN-DYNACTIN MOTOR COMPLEX

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，并确定人类动力蛋白相关疾病的分子基础。动力蛋白是真核细胞微管负端定向转运的主要载体。这一重要运动及其调节蛋白的功能和功能障碍与广泛的细胞功能和人类疾病有关。尽管越来越多的努力来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结构的复杂性。动力蛋白属于AAA+类atp水解机械酶，组装成环状结构，因此，与其他两个细胞骨架运动蛋白家族（运动蛋白和肌球蛋白）相比，具有明显不同的结构特征。动力蛋白也非常大（~1.2 MDa），对动力蛋白的结构-功能研究受到功能性重组动力蛋白的限制。增加动力蛋白的复杂性，动力蛋白与多个辅助链和动力蛋白复合物相关联，所有这些对动力蛋白的几乎每一个细胞功能都是必不可少的。dynactin的最大亚基p150glue（包含dynactin的微管结合结构域）的突变会导致人类的Perry综合征和运动神经元退化。然而，p150glue在动力蛋白功能中的作用尚不清楚。在这项资助中，我们试图通过将超灵敏单分子检测与蛋白质工程相结合来克服这些限制。我们将使用酿酒酵母，重组全长动力蛋白和动力蛋白的唯一来源，来生产这两种多蛋白复合物的稳定野生型和突变型。利用这些生化工具以及多色单分子荧光和光学镊子方法，我们将解决1)动力蛋白的AAA+运动结构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的功能，以及3)人类p150glue突变如何破坏动力蛋白-动力蛋白复合物的功能。这一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治疗干预靶点。

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. PUBLIC HEALTH RELEVANCE: Cytoplasmic dynein is vital to various eukaryotic activities, and mutations in its largest regulatory complex dynactin, cause human neurological disease. We are studying the molecular mechanisms that underlie the function and dysfunction of the dynein-dynactin complex.