Conformational Dynamics in Pin1 REgulation of APP Processing and Abeta Production

描述（由申请人提供）：该提案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胞内结构域（AICD）是该过程的核心参与者。我们实验室的最新研究结果表明，脯氨酰异构酶Pin 1催化APP磷酸化（p）T668 P基序的顺式/反式异构化，并调节APP加工和Abeta产生。我们提出了一套协同实验，以解决的结构和动力学机制，Pin 1调节APP的构象和加工在体外和体内。在目标1中，我们将应用NMR动力学方法来确定结构，微观动力学和热力学，和NH-键动力学参数，描述Pin 1-催化磷酸化AICD。目标1的总体目标是推导出Pin 1中功能运动的基于NMR的机制模型，并预测突变以测试这些模型。在目标2中，我们将制备这些NMR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NMR衍生模型的催化机制和功能运动。在目标3中，我们将随机诱变Pin 1 WW结构域，并选择具有高顺式异构体亲和力的突变体。将在体外和体内测试所选突变体对APP加工和Abeta产生的影响。目的3的总体目标是确定pT 668 P AICD基序的异构体特异性识别在APP加工和Abeta产生中的作用。总的来说，我们将扩展我们有限的基于结构的理解Pin 1功能，探索功能运动通过比较测得的微观和NH-键具体率Pin 1。将使用所有可用技术在体外和体内测试运动模型，包括对APP加工和Abeta产生的影响。通过阐明pT 668-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，这些研究将潜在地为开发新的阿尔茨海默病治疗剂开辟新的途径。用通俗的语言来说，我们最近发现了一种对阿尔茨海默病的发展很重要的新酶。在这项提案中，我们将结合联合收割机核磁共振动力学，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.