Molecular Mechanism of the Parkinson's Disease-associated protein LRRK2

帕金森病相关蛋白LRRK2的分子机制

• 批准号: 10670863
• 负责人: Quyen Quoc Hoang
• 金额: $ 53.24万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10670863
• 关键词: Address; Affect; Alzheimer' s Disease; American; Arginine; Automobile Driving; Back; Binding; Biochemical; Biology; Biomedical Engineering; Biophysics; Cell model; Chemicals; Cryoelectron Microscopy; Data; Disease; Disease Progression; Drug Targeting; Engineering; Enzyme Kinetics; Enzymes; Family; Fingers; GTP Binding; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hydrogen Bonding; Hydrolysis; Impairment; Inflammatory Bowel Diseases; LRRK2 gene; Length; Leprosy; Measures; Methods; Molecular; Molecular Conformation; Molecular Mechanisms of Action; Monitor; Mutation; Nucleotides; Parkinson Disease; Pathogenicity; Phage Display; Phosphotransferases; Process; Property; Regulation; Reporting; Research; Structure; System; Tertiary Protein Structure; Testing; Therapeutic; Therapeutic Intervention; Therapeutic antibodies; Tuberculosis; X-Ray Crystallography; disulfide bond; early phase clinical trial; inhibitor; inorganic phosphate; insight; kinase inhibitor; nervous system disorder; particle; programs; protective allele; reduce symptoms; screening; side effect

Project Summary: LRRK2 is a dual enzyme multidomain protein of unknown function. Mutations in LRRK2 has been associated several different diseases including Parkinson’s disease, inflammatory bowel disease, tuberculosis leprosy, and Alzheimer’s disease. The objective of this research program is to determine the molecular mechanisms of action of LRRK2 by using biochemical, biophysical, and chemical biology methods. We have shown that ROC of LRRK2 is a bona fide GTPase, PD-associated mutations trap it in a persistently activated state by impairing GTPase activity, it undergoes nucleotide-dependent conformational changes that is impaired by disease-associated mutations. The proposed studies seek to understand how these conformational changes in the ROC domain regulate the overall structure and activity of LRRK2. The three aims are: Aim 1: To determine the structures of LRRK2 and its conformational dynamics. a) Determine the structural changes in LRRK2 caused by ROC in different nucleotide-bound states and those carrying PD-associated mutations using cryo-EM. Our preliminary single-particle data reveals 2 conformations which may represent the ‘on’ and ‘off’ states. We will define these structures in the proposed studies. b) Define the atomic structures and interactions of LRRK2 by using X-ray crystallography, which is ideal for defining atoms and bonds. Conditions yielding diffracting crystals of full-length LRRK2 have been identified. c) Define the dynamic interactions of residue R1398 in ROC with the γ-phosphate of GTP using NMR, which is key in regulating mechanism of LRRK2 activity. Aim 2: To define the activities of LRRK2. a) To define the regulation of GTPase activity using measuring GTPase activity of constructs carrying PD- mutations, phosphor-memetic, and engineered conformation-constraining disulfide bonds. b) To determine the kinase active and inactive conformation of LRRK2 by measuring kinase activity of conformation-stabilized constructs. c) To characterize the activity and localization of different conformations of LRRK2 in cell models. Aim 3: To define the conformation-activity relationship of LRRK2. a) To modulate LRRK2 conformation using chemical means by employing high throughput chemical screening and chemical biology methods. b) To engineer biologics and potential therapeutic antibodies for trapping the conformations of LRRK2 by using phage-display screening methods.

项目概要： LRRK2 是一种功能未知的双酶多域蛋白，LRRK2 的突变与此相关。 几种不同的疾病，包括帕金森病、炎症性肠病、结核病、麻风病、 和阿尔茨海默病。 该研究计划的目的是通过使用确定 LRRK2 作用的分子机制 我们已经证明 LRRK2 的 ROC 是真实的。 GTPase，PD 相关突变通过损害 GTPase 活性使其处于持续激活状态， 经历核苷酸依赖性构象变化，该变化受到疾病相关突变的损害。 拟议的研究旨在了解 ROC 结构域中的这些构象变化如何调节 LRRK2 的总体结构和活动的三个目标是： 目标 1：确定 LRRK2 的结构及其构象动力学。 a) 确定ROC在不同核苷酸结合状态下引起的LRRK2结构变化以及那些 使用冷冻电镜携带 PD 相关突变我们的初步单粒子数据揭示了 2 种构象。 它可能代表“开”和“关”状态。我们将在拟议的研究中定义这些结构。 使用 X 射线晶体学研究 LRRK2 的原子结构和相互作用，这是定义原子的理想选择 产生全长 LRRK2 衍射晶体的条件已确定。 使用 NMR 观察 ROC 中残基 R1398 与 GTP 的 γ-磷酸盐的动态相互作用，这是调节的关键 LRRK2 活性的机制。 目标 2：定义 LRRK2 的活动。 a) 通过测量携带PD-的构建体的GTP酶活性来定义GTP酶活性的调节 突变、磷模因和工程化构象约束二硫键 b) 确定 通过测量构象稳定的激酶活性来确定 LRRK2 的激酶活性和失活构象 c) 表征细胞模型中 LRRK2 不同构象的活性和定位。 目标 3：定义 LRRK2 的构象-活性关系。 a) 通过高通量化学筛选，使用化学手段调节 LRRK2 构象 b) 设计生物制剂和潜在的治疗抗体来捕获 使用噬菌体展示筛选方法分析 LRRK2 的构象。

项目成果

Mapping immunological and host receptor binding determinants of SARS-CoV spike protein utilizing the Qubevirus platform.

利用 Qubevirus 平台绘制 SARS-CoV 刺突蛋白的免疫学和宿主受体结合决定因素。

• 发表时间: 2023-12
• 作者: Sanders, Carrie;Dzelamonyuy, Aristide;Ntemafack, Augustin;Alatoom, Nadia;Nchinda, Godwin;Georgiadis, Millie M;Bopda Waffo, Alain
• 通讯作者: Bopda Waffo, Alain

Evolutionary Qβ Phage Displayed Nanotag Library and Peptides for Biosensing.

进化Qβ噬菌体展示纳米标签库和生物传感肽。

• 发表时间: 2023-06-22
• 作者: Ntemafack, Augustin;Dzelamonyuy, Aristide;Nchinda, Godwin;Bopda Waffo, Alain
• 通讯作者: Bopda Waffo, Alain