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）：编码富含亮氨酸的重复激酶2（LRRK2）的新型基因中的突变已被证明是常染色体显性剂，晚期帕金森氏病的最常见原因。然而，对这种多域复合蛋白LRRK2知之甚少，该蛋白具有多个功能上重要的结构域，包括与RAS相关的GTPase结构域和MAPKKK（有丝分裂原激活的蛋白激活蛋白激酶激酶激酶）结构域。遗传研究已经鉴定出GTPase结构域内的两个致病突变，R1441C和MAPKKK域内的G2019。因此，致病性LRK2突变特异性针对的是lrrk2在引起家族性pd中的GTPase和MAPKKK活性。因为GTPase和MAPKKK都位于MAP激酶信号转导级联的上游，LRRRK2可能作为双重调节性的远程疾病，是对临界的远程疾病的相关性，该疾病的态度是量的远程疾病。通过疾病的LRRK2突变介导的致病机制。人神经元细胞系中的结合零件。在特定目的2中，我们的相互作用和附近的MAPKKK域的潜在激活，我们将研究G2019S突变对LRRK2激酶催化，细胞底物的磷酸化的影响此处提出的原则研究也可能导致我们对LRRK2在帕金森氏病发病机理的细胞机制中的致病作用的理解时急需的突破。