Regulation of GTPase activity of LRRK2 and its implication in Parkinson?s disease

描述（由申请人提供）：富含亮氨酸重复激酶2 （LRRK2）是一种新发现的基因，其突变与帕金森病（PD）的晚发性发病机制相关，在家族性和散发性PD中都有前所未有的流行。因此，LRRK2为了解PD的发病机制提供了一个独特的机会。尽管如此，目前对LRRK2的功能及其在PD发病机制中的作用知之甚少。该项目的长期目标是揭示突变体LRRK2参与PD发病机制的分子机制。我们的具体方法是鉴定定义和调节LRRK2的GTPase功能的相互作用蛋白。LRRK2包含多个催化和蛋白质相互作用域，可能参与整合对神经元功能至关重要的信号通路。据报道，LRRK2的Roc结构域具有GTPase活性，可能在分子内调节LRRK2的激酶活性。利用该结构域及其相关的COR结构域作为诱饵，我们最近发现了两种已知的信号蛋白与LRRK2、RacGAP和抗氧化蛋白的特异性相互作用。与RacGAP的相互作用可能有助于定义Roc结构域的功能及其调控。另一方面，LRRK2与抗氧化蛋白的相互作用可能为LRRK2在神经退行性过程（如氧化应激）中的作用提供了分子联系。因此，我们假设LRRK2具有类似rac的GTPase活性，可以调节下游效应物，如抗氧化蛋白，在神经元维持和存活中发挥作用。以下具体目的旨在验证这一假设：(1)检查LRRK2是否具有由已知的相互作用的RacGAP调节的rac样GTPase活性。我们将进一步表征相互作用的特异性，并研究Roc结构域的rac样GTPase活性是否受到GAP的调节。然后我们将确定PD致病突变如何干扰这种调节。最后，我们将研究LRRK2 GTPase活性的调节是否调节其激酶活性。(2)确定LRRK2是否调节一种已知的相互作用的抗氧化蛋白影响神经元存活。我们将首先表征相互作用的特异性，并检查Prx2是否被LRRK2磷酸化。然后，我们将研究LRRK2如何通过这种相互作用调节抗氧化蛋白的功能。我们提出的研究不仅将研究LRRK2在神经元信号传导中的正常调控和功能，还将研究其PD相关突变体在神经元发病机制中的作用。

DESCRIPTION (provided by applicant): Leucine-rich repeat kinase 2 (LRRK2) is a newly identified gene whose mutations are associated with late onset pathogenesis of Parkinson's disease (PD) and have unprecedented prevalence in both familial and sporadic PD. Thus, LRRK2 provides a unique opportunity for understanding the pathogenesis of PD. Despite this, little is currently known about the function of the LRRK2 or its role in PD pathogenesis. The long-term goal of this program is to unveil the molecular mechanisms by which mutant LRRK2 is involved in the pathogenesis of PD. Our specific approach is to identify interacting proteins that define and regulate the GTPase function of LRRK2. LRRK2 contains multiple catalytic and protein interaction domains which may be involved in integrating signaling pathways crucial for neuronal function. The Roc domain of LRRK2 is reported to have GTPase activity that may intra- molecularly regulates the kinase activity of LRRK2. Using this domain and its associated COR domain as bait, we have recently identified specific interactions of two known signaling proteins with LRRK2, a RacGAP and an antioxidant protein. The interaction with a RacGAP may help to define the function of the Roc domain as well as its regulation. On the other hand, the interaction of LRRK2 with an antioxidant protein may provide a molecular link for a role of LRRK2 in neurodegenerative processes such as oxidative stress. Therefore, we hypothesize that LRRK2 has Rac-like GTPase activity that regulates downstream effectors, such as antioxidant proteins, to play a role in neuronal maintenance and survival. The following specific aims are designed to test this hypothesis: (1) To examine whether LRRK2 has Rac-like GTPase activity that is regulated by a known interacting RacGAP. We will further characterize the specificity of the interaction and investigate if the Rac-like GTPase activity of the Roc domain is regulated by the GAP. We will then determine how the PD pathogenic mutations interfere with this regulation. Finally we will examine if the regulation of LRRK2 GTPase activity regulates its kinase activity. (2) To determine whether LRRK2 regulates an identified interacting antioxidant protein to effect neuronal survival. We will first characterize the specificity of the interaction and examine whether Prx2 is phosphorylated by LRRK2. We will then investigate how the function of the antioxidant protein is regulated by LRRK2 through this interaction. Our proposed studies will not only examine the normal regulation and function of LRRK2 in neuronal signaling but also the role of its PD associated mutants in neuronal pathogenesis. PUBLIC HEALTH RELEVANCE: As a newly identified PD associated gene, leucine-rich repeat kinase 2 (LRRK2) shows striking linkage with both familial and sporadic PD and is associated with typical late-onset pathogenesis. Therefore, our proposed studies on the LRRK2 interacting proteins will not only examine the normal regulation and function of LRRK2 in neuronal signaling but also provide insight into the mechanisms by which the pathogenic mutants are involved in PD pathogenesis. Furthermore, functional understanding of LRRK2 interactions with its signaling partners will likely reveal novel therapeutic targets, in addition to LRRK2 itself, for the treatment of PD.