gamma-Secretase: The First Enzymology-Based Study of Intramembrane Proteolysis

γ-分泌酶：第一个基于酶学的膜内蛋白水解研究

• 批准号: 7513409
• 负责人: Daniel R. Dries
• 金额: $ 4.96万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-11-01 至 2010-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7513409
• 关键词: Address; Affect; Age; Aging; Alzheimer' s Disease; Amyloid; Amyloid beta-Protein Precursor; Binding; Biochemistry; Biological Assay; Biological Models; Brain; Catalysis; Cleaved cell; Complex; Condition; Depth; Detection; Disease; Drug Design; Endopeptidases; Enzymatic Biochemistry; Epidemic; Etiology; Fluorescence; Generations; Genetic; Health; Healthcare; Hydrolysis; In Vitro; Individual; Integral Membrane Protein; Ions; Kinetics; Lipids; Liposomes; Longevity; Measures; Medical; Membrane; Metals; Methods; Molecular; Mutation; Numbers; Pathway interactions; Patients; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Population; Positioning Attribute; Post-Translational Protein Processing; Prevalence; Process; Proteins; Proteolysis; Rate; Regulation; Research Proposals; Role; Senile Plaques; Site; Specificity; Testing; Therapeutic; Vesicle; Work; base; cofactor; design; enzyme substrate; familial Alzheimer disease; gamma secretase; in vitro Assay; inhibitor/antagonist; mutant; neurotoxic; nicastrin protein; novel; reconstitution; research study; secretase

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is nearing an epidemic due to a medical paradox: as the general health and lifespan of the population increases, diseases of aging increase in prevalence. Age correlates strongly with AD, affecting 1 in 8 over 65 and nearly 1 in 2 over 85. The startling increase in the number of affected individuals is straining healthcare, particularly as the Baby Boomer generation ages into their 60s and beyond. Yet, few drugs are currently available for treatment or alleviation of AD. The rational design of effective therapeutics can be more efficiently realized with a thorough understanding of the mechanism by which AD manifests itself. At a genetic and cellular level, the roles of the protease gamma-secretase and its substrate, amyloid precursor protein (APP), in the etiology of AD have been known for years. Yet at the molecular level, virtually nothing has been described for the mechanism by which gamma-secretase cleaves APP to give the neurotoxic A-beta peptides. This project specifically addresses three independent aims: 1. The current methods for measuring gamma-secretase activity will be optimized. Various metals, cofactors, and lipids will be screened for their mechanism of modulating gamma-secretase cleavage of substrates in vitro. An existing assay using purified, reconstituted gamma-secretase will be optimized by reconstituting gamma-secretase and its substrates within the same liposome or vesicle. Finally, design of a fluorescence-based assay will allow greater sensitivity and more rapid analysis of activity. 2. The rates and mechanism by which gamma-secretase cleaves its substrates will be elucidated. In vitro assays will be used to determine Km and kcat for various substrates and familial Alzheimer's disease mutants and to characterize inhibitors. Moreover, single-turnover experiments and pre-steady state kinetic methods will be used to elucidate the kinetics and mechanism of each step of gamma-secretase activity. 3. The mechanism by which substrates are differentially recognized and cleaved will be elucidated. Using mutants and binding analysis, the apparent promiscuity of gamma-secretase cleavage will be clarified. Relevance: gamma-secretase is the protein responsible for forming the deadly amyloid-beta plaques found in the brains of Alzheimer's disease patients. This research proposal aims to dissect the individual molecular steps of gamma-secretase activity and the rates at which these steps occur. Such a detailed analysis of the process by which gamma-secretase acts is crucial to the rational design of drugs to alleviate, treat, or ultimately cure Alzheimer's disease.

描述（由申请人提供）：由于医学悖论，阿尔茨海默病（AD）正接近流行：随着人口总体健康状况和寿命的延长，衰老疾病的患病率也随之增加。年龄与 AD 密切相关，65 岁以上的人中有八分之一受到影响，85 岁以上的人有近二分之一受到影响。受影响人数的惊人增加给医疗保健带来了压力，特别是随着婴儿潮一代年龄增长到 60 岁及以上。然而，目前可用于治疗或缓解 AD 的药物很少。通过彻底了解 AD 的表现机制，可以更有效地合理设计有效的治疗方法。在遗传和细胞水平上，蛋白酶γ分泌酶及其底物淀粉样前体蛋白（APP）在AD病因学中的作用早已为人所知。然而，在分子水平上，对于 γ-分泌酶裂解 APP 产生神经毒性 A-β 肽的机制，几乎没有任何描述。该项目具体解决三个独立的目标： 1. 将优化当前测量γ-分泌酶活性的方法。将筛选各种金属、辅因子和脂质在体外调节γ-分泌酶底物裂解的机制。使用纯化的、重构的γ-分泌酶的现有测定将通过在同一脂质体或囊泡内重构γ-分泌酶及其底物来优化。最后，基于荧光的测定的设计将允许更高的灵敏度和更快速的活性分析。 2. 阐明γ-分泌酶裂解其底物的速率和机制。体外测定将用于确定各种底物和家族性阿尔茨海默病突变体的 Km 和 kcat 并表征抑制剂。此外，单周转实验和预稳态动力学方法将用于阐明γ-分泌酶活性每一步的动力学和机制。 3. 阐明差异识别和切割底物的机制。使用突变体和结合分析，γ-分泌酶裂解的明显混杂性将得到澄清。相关性：γ-分泌酶是负责形成阿尔茨海默病患者大脑中致命的β-淀粉样斑块的蛋白质。本研究计划旨在剖析γ-分泌酶活性的各个分子步骤以及这些步骤发生的速率。对γ-分泌酶作用过程的详细分析对于合理设计缓解、治疗或最终治愈阿尔茨海默病的药物至关重要。