Single molecule kinetic studies of gamma-secretase/substrate interaction and the effects of AD-causing mutations

γ-分泌酶/底物相互作用的单分子动力学研究以及 AD 引起的突变的影响

• 批准号: 10323672
• 负责人: Scott Thomas Forth
• 金额: $ 19.27万
• 依托单位: RENSSELAER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-15 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10323672
• 关键词: Active Sites; Alzheimer' s Disease; Amyloid; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Aspartate; Benign; Binding; Biochemical; Biological Assay; Brain; C-terminal; Carboxypeptidase; Catalytic Domain; Complex; Coupled; Dementia; Docking; Dyes; Enzyme Interaction; Enzyme Kinetics; Enzymes; Experimental Designs; Fluorescence; Generations; Homologous Gene; Immobilization; Individual; Integral Membrane Protein; Kinetics; Lipid Bilayers; Liposomes; Measurement; Measures; Mediating; Membrane; Methods; Micelles; Microscope; Monitor; Mutation; Pathologic; Peptide Fragments; Peptide Hydrolases; Process; Production; Proteolysis; Public Health; Senile Plaques; Substrate Interaction; Techniques; Time; Total Internal Reflection Fluorescent; Transmembrane Domain; United States; beta pleated sheet; beta secretase; disease-causing mutation; drug discovery; early onset; enzyme substrate; familial Alzheimer disease; gamma secretase; hydrophilicity; insight; mutant; neurotoxic; novel; presenilin; protein complex; single molecule

Project Summary Amyloid plaque, composed of amyloid-b peptide (Ab), is a pathological hallmark of Alzheimer’s disease (AD). g-secretase is responsible for the cleavage of C99, the C-terminal fragment of 99 residues of amyloid precursor protein (APP), to generate Ab. Previous kinetics studies of g- secretase measured the final production of APP intracellular domain (AICD) and/or Ab. However, the kinetics rates for individual steps of the generation of Ab from C99 are lacking. Here we will use single molecule fluorescence studies to observe enzyme/substrate molecules in real time, and measure the kinetics of enzyme/substrate association and cleavage in g- secretase-mediated intramembrane proteolysis to generate Ab (Aim1), and determine how familial AD (FAD) mutations alter the kinetics of enzyme/substrate association and cleavage in AD (Aim2).

项目摘要 由淀粉样蛋白b肽（Ab）组成的淀粉样蛋白斑块是阿尔茨海默病的病理标志 疾病（AD）。g-分泌酶负责切割C99（99的C末端片段） 淀粉样前体蛋白（APP）残基，以产生Ab。以前的动力学研究g- 分泌酶测量APP胞内结构域（AICD）和/或Ab的最终产生。 然而，缺乏从C99产生Ab的各个步骤的动力学速率。 在这里，我们将使用单分子荧光研究来观察酶/底物分子 在真实的时间，并测量酶/底物缔合和裂解的动力学，以g- 分泌酶介导的膜内蛋白水解产生Ab（Aim 1），并确定如何 家族性AD（FAD）突变改变了酶/底物结合和切割的动力学， AD（Aim 2）。