Molecular Dissection of Cytoplasmic Dynein

细胞质动力蛋白的分子解剖

• 批准号: 6702245
• 负责人: SAMARA L RECK-PETERSON
• 金额: $ 4.89万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6702245
• 关键词: Saccharomyces cerevisiae; adenosine triphosphate; binding sites; biomechanics; cell motility; dynein ATPase; fungal proteins; gene deletion mutation; hydrolysis; immunoprecipitation; molecular site; postdoctoral investigator; protein protein interaction; protein purification; protein structure function

DESCRIPTION (provided by applicant): The goal of this project is to characterize the mechanochemical properties of the molecular motor dynein using a structure- function approach in the model system, S. cerevisiae. This work is relevant to human disease because humans lacking functional axonemal dynein develop Kartagner syndrome, a disease resulting in chronic respiratory problems, infertility and mirror- image organ positioning. Cytoplasmic dynein is thought to be present in all eukaryotic cells and has multiple, essential roles in cell growth and division. Research in the dynein field has been hampered by the lack of a system in which mutant proteins can be made and analyzed in vitro. The first aim of this project is to develop in vitro assays to study the motile properties of affinity purified yeast cytoplasmic dynein. Such biophysical assays have been pioneered by the Vale lab and others. These assays will be used to test hypotheses described in the following two aims. Unlike other cytoskeletal molecular motors, dynein contains multiple nucleotide binding sites that may bind and hydrolyze ATP to produce the force that drives movement. The second aim of this project is to determine the relative contribution of each ATP binding site in dynein function by mutating conserved residues predicted to be involved in ATP hydrolysis. Finally, the role of dynein-associated proteins in dynein-mediated motility will be analyzed. One such associated protein is the Liscencephaly 1 protein, which, when defective causes a severe brain developmental disease in humans. As dyneins are evolutionarily distinct from the molecular motors kinesin and myosin, these studies represent a frontier in the motor field and may reveal novel mechanisms for motor protein function.

描述（由申请人提供）： 该项目的目的是使用模型系统中的结构函数方法（酿酒酵母中的结构功能方法）表征分子运动蛋白的机械化学特性。这项工作与人类疾病有关，因为缺乏功能性轴突动力蛋白的人会发展为Kartagner综合征，这种疾病导致慢性呼吸疾病，不育和镜像器官定位。胞质动力蛋白被认为存在于所有真核细胞中，并且在细胞生长和分裂中具有多个基本作用。由于缺乏可以在体外进行突变蛋白的系统，在动力蛋白领域的研究受到了阻碍。该项目的第一个目的是开发体外测定，以研究亲和力纯化酵母菌细胞质动力蛋白的运动特性。这种生物物理测定已由谷实验室和其他实验室开创。这些测定将用于测试以下两个目标中描述的假设。与其他细胞骨架分子电机不同，动力蛋白包含多个可以结合并水解ATP的核苷酸结合位点，以产生驱动运动的力。该项目的第二个目的是通过突变预测参与ATP水解的保守残基来确定Dynein功能中每个ATP结合位点的相对贡献。最后，将分析与动力蛋白相关蛋白在动力蛋白介导的运动中的作用。这种相关的蛋白质是liscencephaly 1蛋白，当有缺陷引起人类严重的脑发育疾病时。由于动力蛋白在进化上与分子电动机动力素和肌球蛋白不同，因此这些研究代表了运动场中的前沿，并且可能揭示了运动蛋白功能的新型机制。