Structure-Activity Analysis of LRRK2 Associated:PD

描述（由申请人提供）：编码富亮氨酸重复激酶2（LRRK 2）的新基因突变最近被证明是常染色体显性遗传迟发性帕金森病的最常见原因。然而，对这种多结构域复合蛋白LRRK 2知之甚少，LRRK 2具有几个功能重要的结构域，包括Ras相关的GT3结构域和MAPKKK（促分裂原活化蛋白激酶）结构域。遗传学研究已经确定了两个致病性突变，GTdR结构域内的R1441 C和MAPKKK结构域内的G2019 S。因此，致病性LRRK 2突变特异性地靶向LRRK 2的GTTT和MAPKKK活性，从而导致家族性PD。由于GT3和MAPKKK均位于MAP激酶信号转导级联的上游，LRRK 2可能充当控制与帕金森病发病机制相关的关键细胞过程的双重调节开关。我们的长期目标是阐明致病LRRK 2突变介导的致病机制。在本申请中，我们提出研究R1441 C和G2019 S突变与LRRK 2的催化活性之间的结构-活性关系。两个具体的目的是集中在分析的致病性突变对GT3和MAPKKK活动的LRRK 2，其相关的底物和结合伙伴在人类神经元细胞系的影响。将建立表达FLAG表位标记的LRRK 2的转染细胞系，所述LRRK 2在各自的GT3和MAPKKK结构域内携带R1441 C和G2019 S突变。在具体目标1中，我们将研究R1441 C突变对LRRK 2 GTdR活性的影响，包括鸟嘌呤核苷酸结合和水解、辅因子相互作用以及附近MAPKKK结构域的潜在激活。在特定目标2中，我们将研究G2019 S突变对LRRK 2激酶催化、细胞底物磷酸化和已知MAP激酶通路活化的影响。这些拟议的实验可能会产生必要的数据，为启动疾病过程中的致病性LRRK 2突变提供结构基础。这里提出的原理验证研究也可能导致我们对LRRK 2在帕金森病发病机制的细胞机制中的致病作用的理解急需突破。

DESCRIPTION (provided by applicant): Mutations in a novel gene encoding the leucine-rich repeat kinase 2 (LRRK2) have recently been shown to be the most common cause of autosomal dominant, late-onset Parkinson's disease. However, little is known about this multi-domain complex protein, LRRK2, that has several functionally important domains, including a Ras-related GTPase domain and a MAPKKK (mitogen- activated protein kinase kinase kinase) domain. Genetic studies have identified two pathogenic mutations, R1441C within the GTPase domain, and G2019S within the MAPKKK domain. Therefore, pathogenic LRRK2 mutations specifically target the GTPase "and MAPKKK activities of LRRK2 in causing familial PD. As both the GTPase and MAPKKK are located upstream of MAP kinase signal transduction cascades, LRRK2 is likely to serve as a dual regulatory switch controlling critical cellular processes relevant to the pathogenesis of Parkinson's disease. Our long-term objective is to elucidate the pathogenic mechanisms mediated by the disease-causing LRRK2 mutations. In this application, we propose to investigate the structure-activity relationship between the R1441C and G2019S mutations and the catalytic activities of LRRK2. Two specific aims are designed to focus on the analyses of the effects of the pathogenic mutations on GTPase and MAPKKK activities of LRRK2, and their associated substrates and binding-partners in human neuronal cell lines. Transfected cell lines expressing FLAG epitope-tagged LRRK2 carrying the R1441C and G2019S mutations within the respective GTPase and MAPKKK domains will be established. In Specific Aim 1, we will examine the effect of R1441C mutation on LRRK2 GTPase activity in terms of guanine nucleotide binding and hydrolysis, co-factor interactions, and potential activation of nearby MAPKKK domain. In Specific Aim 2, we will investigate the effect of G2019S mutation on LRRK2 kinase catalysis, phosphorylation of cellular substrates, and activation of known MAP kinase pathways. These proposed experiments will likely yield essential data that provide a structural basis for the pathogenic LRRK2 mutations in initiating the disease process. The proof-of- principle research proposed here is also likely to lead to a much-needed breakthrough in our understanding of the pathogenic roles of LRRK2 in the cellular mechanisms underlying the pathogenesis of Parkinson's disease.