Cell Biology of Presenilin 1 and Associated Proteins

描述（由申请人提供）：本项目旨在澄清 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产生中起关键作用。 然而，PS1复合物的分子组成尚未阐明。 在这方面，一种称为nicastrin的I型膜蛋白最近被发现。 鉴定为PS1复合物的化学计量组分。实验 nicastrin的突变体影响AB的产生和Notch 1的切割，这表明 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 nicastrin的分布，nicastrin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nicastrin的生物化学共分布， 免疫细胞化学方法。除了在调解方面的作用外， APP和Notch，PS1膜内γ-分泌酶裂解也是已知的 调节几种膜蛋白，包括APP和 神经营养因子受体TrkB。初步结果显示， 影响APP加工的实验性PS1突变体也改变了亚细胞内 APP、APP CTF、APLP 2和TrkC的分布。目标2下的研究将 扩大这些调查，以审查PS1在贩运人口方面的作用， APP，APP同系物，神经营养因子受体，低密度脂蛋白受体相关 蛋白质（LRP）、转铁蛋白受体和AMPA受体。根据目标3， 我们概述了生物化学策略，以确定和表征分子 PS1复合物的组分和我们表征γ-分泌酶的方法 体外活性这些研究也将有助于 γ-分泌酶新底物鉴定统称 建议的研究将提供新的见解： nicastrin与PS1在γ-分泌酶调节中的关系 活动; PS1内包含的组件的标识-驻留高 分子量复合物;以及PS1在调节 细胞内运输和膜蛋白的代谢。

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.