Structure-Activity Analysis of LRRK2 Associated:PD

LRRK2相关:PD的构效分析

• 批准号: 7135478
• 负责人: SHU G. CHEN
• 金额: $ 19万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7135478
• 关键词: Parkinson' s disease; SDS polyacrylamide gel electrophoresis; biological signal transduction; cell line; enzyme activity; enzyme structure; enzyme substrate complex; gene mutation; guanine nucleotide binding protein; guanosinetriphosphatases; high performance liquid chromatography; intermolecular interaction; mitogen activated protein kinase; molecular pathology; radiotracer; serine threonine protein kinase; thin layer chromatography

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.

描述（由申请人提供）：编码富含亮氨酸重复激酶 2 (LRRK2) 的新基因的突变最近已被证明是常染色体显性迟发性帕金森病的最常见原因。然而，人们对这种多结构域复合蛋白 LRRK2 知之甚少，该蛋白具有多个功能上重要的结构域，包括 Ras 相关的 GTPase 结构域和 MAPKKK（丝裂原激活蛋白激酶激酶）结构域。遗传学研究已鉴定出两种致病性突变：GTPase 结构域内的 R1441C 和 MAPKKK 结构域内的 G2019S。因此，致病性 LRRK2 突变特异性靶向 LRRK2 的 GTPase 和 MAPKKK 活性，导致家族性 PD。由于 GTPase 和 MAPKKK 均位于 MAP 激酶信号转导级联的上游，LRRK2 很可能充当控制关键细胞过程的双重调节开关。我们的长期目标是阐明引起疾病的 LRRK2 突变介导的致病机制。在本申请中，我们建议研究 R1441C 和 G2019S 突变与 LRRK2 催化活性之间的结构 - 活性关系，旨在重点分析致病性突变对 LRRK2 的 GTPase 和 MAPKKK 活性的影响，及其在人神经元细胞系中的相关底物和结合伴侣，表达带有 R1441C 和 R1441C 的 FLAG 表位标记的 LRRK2 的转染细胞系。将确定各自 GTPase 和 MAPKKK 结构域内的 G2019S 突变。在具体目标 1 中，我们将检查 R1441C 突变对 LRRK2 GTPase 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。在具体目标 2 中，我们将研究 G2019S 突变对 LRRK2 激酶催化、细胞底物磷酸化以及已知 MAP 激酶途径激活的影响。这些拟议的实验可能会产生重要的数据，为引发疾病过程的致病性 LRRK2 突变提供结构基础。这里提出的原理验证研究也可能为我们对 LRRK2 在帕金森病发病机制的细胞机制中的致病作用的理解带来急需的突破。