Structure and Function of the Parkinson's disease associated protein LRRK2

 描述(由申请人提供)：在这里，我们试图了解LRRK2结构域之间的结构和相互作用如何调节其活性，以及帕金森氏病相关突变如何影响这些过程。这些研究领域意义重大，因为LRRK2在开发有效治疗帕金森病(PD)方面有着最大的希望，而目前还没有治疗帕金森病的方法。LRRK2的突变导致疾病明显扰乱LRRK2的激酶活性；然而，这样做的机制是复杂的，因为远离激活域的突变部位也对激酶活性产生类似的影响。解开这些机制的主要障碍是缺乏可用于详细的生化和生物物理研究的蛋白质样本。我们已经克服了这一点，开发了产生高度纯化的样本的程序，这些样本适合进行详细研究。在这里，我们利用这些样本来研究LRRK2的结构和功能，梳理其活性是如何调节的，并确定其在疾病发病机制中的作用机制。本项目拟研究的三个方面是：1.确定PD相关突变对LRRK2 ROC结构域结构和功能的影响。我们已经证明，PD突变R1441H损害了LRRK2的ROC结构域的GTP酶活性。这种损伤是通过扭曲活性中心、改变开关区的构象或阻碍镁离子的结合而发生的。2.确定ROC的GTP酶激活结构域，为GTP酶的调控奠定结构基础。我们已经证明了LRRK2的ROC结构域具有低的固有GTPase活性，并且我们已经看到它可以被LRRK2中的一个未知结构域激活30倍。我们将使用生化和结构研究相结合的方法来识别和定义这种激活。 机制。3.定义LRRK2内结构域之间的全球结构相互作用。在LRRK2的不同部分都发现了突变，但它们都会影响距离突变位置几个区域的激酶活性，从而表明这些区域相互作用，协同发挥共同的功能。我们将绘制LRRK2中所有域的空间排列图，以深入了解它们如何协同工作。

 DESCRIPTION (provided by applicant): Here we seek to understand how structure and interactions between the domains of LRRK2 regulate its activities, and also how Parkinson's disease-associated mutations affect these processes. Those areas of study are significant because LRRK2 holds the strongest promise for developing effective treatments for Parkinson's disease (PD), for which there is currently none. Mutations in LRRK2 leading to disease clearly perturb LRRK2 kinase activity; however, the mechanism in doing so is complex as mutation sites remote from the kinase domain also exert similar effects on kinase activity. The major roadblock in unraveling these mechanisms had been the lack of protein samples amenable for detail biochemical and biophysical studies. We have overcome that by developing procedures that yield highly purified samples amenable for detail studies. Here we use these samples to study the structure and function of LRRK2, to tease out how its activities are regulated, and to define its mechanism in disease pathogenesis. The three areas to be investigated in this project are: 1. To determine the effects of PD-associated mutations on the structure and function of the Roc domain of LRRK2. We have shown that the PD-mutation R1441H impairs GTPase activity of the Roc domain of LRRK2. This impairment occurs through distorting the active-site, altering the conformation of the switch regions, or impeding the binding of magnesium ions. 2. To determine the GTPase activation domain of Roc and to define the structural basis for GTPase regulation. We have shown that the Roc domain of LRRK2 has low intrinsic GTPase activity, and we have seen that it could be activated 30-fold by an unknown domain within LRRK2. We will use a combination of biochemical and structural studies to identify and define this activation mechanism. 3. To define global structural interactions between the domains within LRRK2. Mutations are found in various parts of LRRK2, but they all affect kinase activity which can be several domains away from the mutation sites, thus indicating that these domains interact with one another working in concert to exert a common function. We will map the spatial arrangement of all domains within LRRK2 to gain insight into how they work together.