Molecular Dissection of Cytoplasmic Dynein

描述(由申请人提供)： 本项目的目标是在酿酒酵母模型系统中使用结构-功能方法来表征分子马达动力蛋白的机械力化学性质。这项工作与人类疾病相关，因为缺乏功能性轴线动力蛋白的人类会患上卡塔格纳综合征，这种疾病会导致慢性呼吸问题、不孕不育和镜像器官定位。细胞质动力蛋白被认为存在于所有真核细胞中，并在细胞生长和分裂中发挥多种重要作用。动力蛋白领域的研究一直受到缺乏能够在体外制造和分析突变蛋白的系统的阻碍。本项目的第一个目的是建立体外分析方法来研究亲和纯化的酵母胞质动力蛋白的运动特性。这样的生物物理分析是由淡水河谷实验室和其他实验室开创的。这些分析将用于检验以下两个目标中描述的假设。与其他细胞骨架分子马达不同，动力蛋白含有多个核苷酸结合位点，可以结合和水解三磷酸腺苷，产生驱动运动的力。该项目的第二个目标是通过突变预计参与ATP水解的保守残基来确定每个ATP结合位点在动力蛋白功能中的相对贡献。最后，将分析动力蛋白相关蛋白在动力蛋白介导的运动中的作用。其中一种相关的蛋白质是李氏脑蛋白1，当缺陷时，它会导致人类严重的大脑发育疾病。由于动力蛋白在进化上不同于运动蛋白和肌球蛋白的分子马达，这些研究代表了运动领域的前沿，并可能揭示运动蛋白功能的新机制。

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.