Cell Biology of Presenilin 1 and Associated Proteins

早老素 1 和相关蛋白的细胞生物学

• 批准号: 6755908
• 负责人: GOPAL THINAKARAN
• 金额: $ 32.63万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6755908
• 关键词: amyloid proteins; biochemistry; cell biology; cell line; endopeptidases; enzyme activity; genetically modified animals; glycoproteins; immunoaffinity chromatography; immunocytochemistry; mass spectrometry; membrane proteins; presenilin; protein biosynthesis; protein localization; protein protein interaction; protein transport

DESCRIPTION (provided by applicant): This project seeks to clarify the molecular and cellular mechanisms by which Presenilins (PS1 and PS2) and associated molecules mediate protein trafficking, gamma-secretase activity and AB production. Recent studies have revealed that high molecular weight protein complexes containing PS play a critical role in AB production. However, the molecular composition of PS1 complexes has not been elucidated. In this regard, a type I membrane protein, termed nicastrin, was recently identified as a stoichiometric component of the PS1 complex. Experimental mutants of nicastrin effect AB production and Notch 1 cleavage, suggesting an accessory role for this molecule in regulating gamma secretase activity. Under Aim 1, we propose to characterize the cellular and subcellular distributions of nicastrin, the interaction of nicastrin and PS1 using deletion mutagenesis strategies, and assessment of the role of PS1-nicastrin interactions on gamma-secretase activity. Finally, we will investigate the co-distribution of APP, APP B-CTF, PS1 and nicastrin by biochemical and immunocytochemical methods. In addition to its role in mediating intramembranous gamma-secretase cleavage of APP and Notch, PS1 is also known to regulate the trafficking of several membrane proteins, including APP and the neurotrophin receptor, TrkB. Our Preliminary Results show that experimental PS1 mutants that effect APP processing also alter the subcellular distributions of APP, APP CTFs, APLP2 and TrkC. Studies under Aim 2 will extend these investigations to examine the role of PS1 on the trafficking of APP, APP homologues, neurotrophin receptors, low-density lipoprotein receptor-related protein (LRP), transferrin receptor, and AMPA receptors. Under Aim 3, we outline biochemical strategies to identify and characterize the molecular components of PS1 complexes and our approaches to characterize gamma-secretase activity in vitro. These latter studies will also facilitate the identification of novel substrates of gamma-secretase. Collectively, the studies proposed will provide new insights into: the functional relationship between nicastrin and PS1 in modulation of gamma-secretase activity; the identity of components contained within PS1-resident high molecular weight complexes; and the function of PS1 in regulating intracellular trafficking and metabolism of membrane proteins.

描述（由申请人提供）：该项目旨在澄清 早老素（PS1 和 PS2）和 相关分子介导蛋白质运输、γ-分泌酶活性和 AB生产。最近的研究表明，高分子量 含有 PS 的蛋白质复合物在 AB 生产中起着至关重要的作用。 然而，PS1复合物的分子组成尚未阐明。 在这方面，最近发现了一种 I 型膜蛋白，称为尼卡斯特林 (nicastrin)。 被鉴定为 PS1 复合物的化学计量成分。实验性的 Nicastrin 的突变体影响 AB 的产生和 Notch 1 的切割，表明 该分子在调节γ分泌酶活性中的辅助作用。 在目标 1 下，我们建议表征细胞和亚细胞 尼卡斯特林的分布、尼卡斯特林和 PS1 的相互作用 缺失诱变策略以及 PS1-nicastrin 作用的评估 γ-分泌酶活性的相互作用。最后，我们将调查 APP、APP B-CTF、PS1 和尼卡斯特林通过生化和 免疫细胞化学方法。除了其调解作用外 APP 和 Notch、PS1 的膜内 γ 分泌酶裂解也是已知的 调节多种膜蛋白的运输，包括 APP 和 神经营养蛋白受体 TrkB。我们的初步结果表明 影响 APP 处理的实验性 PS1 突变体也会改变亚细胞 APP、APP CTF、APLP2 和 TrkC 的分布。目标 2 下的研究将 扩大这些调查范围，以检验 PS1 在贩运人口中的作用 APP、APP 同系物、神经营养素受体、低密度脂蛋白受体相关 蛋白 (LRP)、转铁蛋白受体和 AMPA 受体。在目标 3 下， 我们概述了识别和表征分子的生化策略 PS1 复合物的成分以及我们表征 γ 分泌酶的方法 体外活性。后面这些研究也将有助于 γ-分泌酶新底物的鉴定。总的来说， 拟议的研究将为以下方面提供新的见解： 尼卡斯特林和 PS1 在调节 γ 分泌酶中的关系 活动; PS1-resident high 中包含的组件的身份 分子量复合物；以及PS1的调节功能 膜蛋白的细胞内运输和代谢。