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

描述(申请人提供)：由于一个医学悖论：阿尔茨海默病(AD)正接近流行：随着人口总体健康和寿命的增加，老年病的患病率增加。年龄与阿尔茨海默病密切相关，65岁以上的人中有八分之一，85岁以上的人中有近二分之一。受影响人数的惊人增长给医疗保健带来了压力，特别是在婴儿潮一代进入60岁及以上的情况下。然而，目前治疗或缓解AD的药物很少。深入了解阿尔茨海默病的发病机制，才能更有效地合理设计有效的治疗方法。在遗传和细胞水平上，蛋白水解酶及其底物--淀粉样前体蛋白(APP)在阿尔茨海默病的病因中的作用早已为人所知。然而，在分子水平上，几乎没有关于伽马分泌酶裂解APP产生神经毒性A-β多肽的机制的描述。该项目具体针对三个独立的目标：1.将优化目前测量伽马分泌酶活性的方法。各种金属、辅因子和脂类将在体外筛选它们对底物的伽马分泌酶裂解的调节机制。现有的使用纯化的、重组的伽马分泌酶的检测将通过在相同的脂质体或囊泡中重组伽玛分泌酶及其底物来进行优化。最后，设计一种基于荧光的分析将允许更高的灵敏度和更快的活性分析。2.将阐明伽玛分泌酶裂解底物的速率和机制。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，并确定抑制剂的特性。此外，还将使用单周转实验和稳态前动力学方法来阐明伽玛分泌酶活性的每一步的动力学和机理。3.底物的差异识别和切割的机制将被阐明。利用突变体和结合分析，将澄清伽马分泌酶切割的明显混杂。相关性：伽马分泌酶是一种蛋白质，负责在阿尔茨海默病患者的大脑中发现致命的淀粉样β-斑块。这项研究计划旨在剖析伽马分泌酶活性的各个分子步骤以及这些步骤发生的速率。对伽马分泌酶的作用过程进行如此详细的分析，对于合理设计减轻、治疗或最终治愈阿尔茨海默病的药物至关重要。

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.