Structure-Activity Analysis of LRRK2 Associated:PD

描述(由申请人提供)：编码富含亮氨酸的重复蛋白激酶2(LRRK2)的一种新基因的突变最近被证明是常染色体显性遗传的迟发性帕金森病的最常见原因。然而，人们对这种多结构域的复杂蛋白LRRK2知之甚少，它具有几个重要的功能结构域，包括一个与RAS相关的GTPase结构域和一个MAPKKK(丝裂原激活的蛋白激酶激酶)结构域。遗传学研究已经确定了两个致病突变，R1441C位于GTPase区域，G2019S位于MAPKKK区域。因此，致病的LRRK2突变特异性地针对LRRK2的GTPase和MAPKKK活性而导致家族性帕金森病。由于GTPase和MAPKKK都位于MAP激酶信号转导通路的上游，LRRK2很可能是一个双重调控开关，控制着与帕金森病发病相关的关键细胞过程。我们的长期目标是阐明致病基因LRRK2突变所介导的致病机制。在这一应用中，我们建议研究R1441C和G2019S突变之间的构效关系和LRRK2的催化活性。为了分析致病突变对LRRK2及其相关底物和结合伙伴的GTP酶和MAPKKK活性的影响，我们设计了两个特定的目标。将建立在各自的GTPase和MAPKKK结构域中表达带有标志表位标签的LRRK2的转基因细胞系，该细胞系携带R1441C和G2019S突变。在具体目标1中，我们将从鸟核苷酸结合和水解、辅因子相互作用以及附近MAPKKK结构域的潜在激活等方面来研究R1441C突变对LRRK2 GTPase活性的影响。在特定的目标2中，我们将研究G2019S突变对LRRK2激酶催化、细胞底物的磷酸化和已知MAP激酶通路激活的影响。这些拟议的实验可能会产生必要的数据，为启动疾病过程的致病LRRK2突变提供结构基础。这里提出的原则证明研究也可能在我们对LRRK2在帕金森氏病发病机制的细胞机制中的致病作用的理解方面取得亟需的突破。

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.