Conformational Dynamics in Pin1 REgulation of APP Processing and Abeta Production

描述(由申请人提供)：这项建议建立在大量文献的基础上，这些文献表明淀粉样前体蛋白(APP)加工和Abeta产生的异常调节是阿尔茨海默病的主要原因，APP细胞内域(AICD)在这一过程中发挥核心作用。我们实验室最近的研究结果表明，Prolyl异构酶Pin1催化APP的(P)T668P基序的顺式/反式异构化，并调节APP的加工和Abeta的产生。我们提出了一系列协同实验来研究Pin1在体内外调控APP构象和加工的结构和动力学机制。在目标1中，我们将应用核磁共振动力学方法来确定描述磷酸化AICD的Pin1催化的结构、微观动力学和热力学以及NH键动力学参数。目标1的总体目标是为Pin1中的功能运动推导基于核磁共振的机制模型，并预测突变以测试这些模型。在目标2中，我们将制造这些核磁共振预测的突变体，并评估它们在体外和体内对APP加工和Abeta产生的影响。目标2的总体目标是验证用于催化机制和功能运动的核磁共振衍生模型。在目标3中，我们将随机突变Pin1的WW结构域，并选择具有高顺式异构体亲和力的突变体。选定的突变体对APP加工和Abeta产生的影响将在体外和体内进行测试。目标3的总体目标是确定pT668P AICD基序的异构体特异性识别在APP加工和Abeta生产中的作用(S)。总体而言，我们将扩展我们对Pin1功能的有限结构理解，通过比较Pin1中测量的微观和NH键比速率来探索功能运动。运动模型将使用所有可用的技术在体外和体内进行测试，包括对APP处理和Abeta产生的影响。通过阐明pT668-APP的顺式和反式异构体的调节，并确定它们在APP加工和Abeta产生中的作用，这些研究可能会为开发新的阿尔茨海默病疗法开辟新的途径。在通俗易懂的语言中，我们最近发现了一种对阿尔茨海默病的发展至关重要的新酶。在这项提案中，我们将结合核磁共振动力学、细胞和分子生物学方法来研究这种酶如何影响阿尔茨海默病的过程，并希望最终确定新的治疗靶点。

DESCRIPTION (provided by applicant): This proposal builds on extensive literature that implicates aberrant regulation of amyloid precursor protein (APP) processing and Abeta production as a major cause of Alzheimer's disease, and the APP intracellular domain (AICD) as a central player in this process. Recent results from our labs show that the prolyl isomerase Pin1 catalyzes cis/trans isomerization of the phosphorylated (p) T668P motif of APP and regulates APP processing and Abeta production. We propose a set of synergistic experiments to address the structural and dynamic mechanisms by which Pin1 regulates the conformation and processing of APP in vitro and in vivo. In Aim 1 we will apply NMR dynamics methods to determine the structural, microscopic kinetic and thermodynamic, and NH-bond dynamic parameters that describe Pin1-catalysis of phosphorylated AICD. The overall goal of Aim 1 is to derive NMR-based mechanistic models for functional motions in Pin1, and to predict mutations to test these models. In Aim 2 we will make these NMR-predicted mutants and evaluate their effects on APP processing and Abeta production in vitro and in vivo. The overall goal of Aim 2 is to validate NMR-derived models for the catalytic mechanism and for functional motions. In Aim 3 we will randomly mutagenize the Pin1 WW domain and select for mutants with high cis-isomer affinity. The effects of the selected mutants on APP processing and Abeta production will be tested in vitro and in vivo. The overall goal of Aim 3 is to determine the role(s) of isomer-specific recognition of the pT668P AICD motif in APP processing and Abeta production. Overall, we will extend our limited structure-based understanding of Pin1 function, exploring functional motions by comparing measured microscopic and NH- bond specific rates in Pin1. Motional models will be tested in vitro and in vivo using all available technology, including the effects on APP processing and Abeta production. By elucidating the regulation of the cis and trans isomers of pT668-APP and establishing their roles in APP processing and Abeta production, these studies will potentially open new avenues for development of novel Alzheimer's disease therapeutics. In lay language, we have recently identified a new enzyme important for the development of Alzheimer's disease. In this proposal, we will combine NMR dynamics, cellular and molecular biology approaches to study how this enzyme affects Alzheimer's disease processes and hope to eventually identify new therapeutic targets.