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

描述（由申请人提供）：阿尔茨海默病（AD）由于医学悖论而接近流行病：随着人口的总体健康和寿命的增加，衰老疾病的患病率增加。年龄与AD密切相关，65岁以上的患者占1/8，85岁以上的患者占近1/2。受影响人数的惊人增加正在使医疗保健紧张，特别是随着婴儿潮一代年龄增长到60多岁及以上。然而，目前很少有药物可用于治疗或缓解AD。有效治疗的合理设计可以更有效地实现与AD表现出自己的机制的透彻理解。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病因学中的作用多年来一直为人所知。然而，在分子水平上，几乎没有任何关于γ-分泌酶切割APP以产生神经毒性A-β肽的机制的描述。该项目具体涉及三个独立的目标：1。目前用于测量γ-分泌酶活性的方法将被优化。将筛选各种金属、辅因子和脂质在体外调节γ-分泌酶切割底物的机制。将通过在相同脂质体或囊泡内重构γ-分泌酶及其底物来优化使用纯化的重构γ-分泌酶的现有测定。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.γ-分泌酶切割其底物的速率和机制将被阐明。体外试验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，单周转实验和前稳态动力学方法将用于阐明γ-分泌酶活性的每一步的动力学和机制。3.将阐明底物被差异识别和裂解的机制。使用突变体和结合分析，γ-分泌酶切割的表观混杂性将得到澄清。相关性：γ-分泌酶是一种蛋白质，在阿尔茨海默病患者的大脑中发现，它负责形成致命的β-淀粉样蛋白斑块。这项研究计划旨在剖析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药物以减轻、治疗或最终治愈阿尔茨海默病至关重要。

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.