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-动力肌动蛋白运动复合体的分子机制，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中微管负末端定向运输的主要载体。这一重要运动及其调节蛋白的功能和功能障碍有助于广泛的细胞功能和人类疾病。尽管越来越多的努力来定义动力蛋白的功能特性，动力蛋白的机械化学的分子机制仍然知之甚少。这种缺陷主要源于动力蛋白的结构复杂性。动力蛋白属于组装成环状结构的ATP水解机械酶的AAA+类，因此，与其他两种细胞骨架运动蛋白家族驱动蛋白和肌球蛋白相比，动力蛋白具有特征性不同的结构特征。动力蛋白也是非常大的（~1.2 MDa）和动力蛋白的结构-功能研究受到功能性重组动力蛋白的可用性的限制。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，所有这些对于动力蛋白的几乎所有细胞功能都是必不可少的。动力肌动蛋白最大的亚基p150 glued含有动力肌动蛋白的微管结合域，它的突变导致人类佩里综合征和运动神经元变性。然而，p150 glued在动力蛋白功能中的作用仍然未知。在这项研究中，我们试图通过将超灵敏的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S。酿酒酵母，重组全长动力蛋白和动力肌动蛋白的唯一来源，以产生两种多蛋白复合物的稳定野生型和突变体形式。利用这些生物化学工具和单分子荧光和光镊方法，我们将解决1）动力蛋白的AAA+马达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功能，以及3）人类p150胶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，并可能确定动力蛋白-动力蛋白复合物中的治疗干预靶点。

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.