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

描述（由申请人提供）：由于医学悖论，阿尔茨海默病（AD）接近流行病：随着人口总体健康和寿命的增加，老年疾病的患病率增加。年龄与阿尔茨海默病密切相关，65岁以上有八分之一，85岁以上有近二分之一。受影响人数的惊人增长正在给医疗保健带来压力，尤其是在婴儿潮一代步入60多岁甚至更老的时候。然而，目前很少有药物可用于治疗或减轻阿尔茨海默病。只有深入了解阿尔茨海默病的表现机制，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，蛋白酶-分泌酶及其底物淀粉样前体蛋白（APP）在阿尔茨海默病病因学中的作用已被了解多年。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-肽的机制的描述。该项目具体解决三个独立的目标：1。目前测量γ -分泌酶活性的方法将得到优化。各种金属，辅助因子和脂质将筛选其调节γ -分泌酶切割底物的体外机制。现有的使用纯化的重组γ -分泌酶的实验将通过在相同的脂质体或囊泡内重组γ -分泌酶及其底物来优化。最后，设计一种基于荧光的检测方法将允许更高的灵敏度和更快速的活性分析。2. 伽马分泌酶裂解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，并确定抑制剂的特征。此外，将采用单次翻转实验和稳态前动力学方法来阐明γ -分泌酶活性各步骤的动力学和机制。3. 底物被区分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γ分泌酶切割的明显混杂性。相关性：γ分泌酶是在阿尔茨海默病患者的大脑中发现的负责形成致命淀粉样斑块的蛋白质。本研究计划旨在剖析γ -分泌酶活性的单个分子步骤和这些步骤发生的速率。这种对分泌酶作用过程的详细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.