GAMMA SECRETASE ACTIVITY N COORDINATED CELL-CELL INTERACTIONS

PSL/γ-分泌酶是一种主要由负责其底物膜内切割的膜蛋白组成的大型酶复合体。PS1/γ-分泌酶最初被证明可以裂解淀粉样前体蛋白(APP)和Notch，分别释放Abeta多肽和Notch胞内结构域。我们和其他人最近已经确定了更多的底物正在进行依赖于PS1的蛋白降解过程。尽管这些底物具有不同的功能，但我们将探索PS1/伽马-分泌酶复合体在Abeta生成和细胞-细胞连接中所起作用的潜在关系。我们最近发现了一种新的PS1/伽马分泌酶活性底物Nectin-1，它是一种参与突触形成的黏附连接蛋白，目前我们正在鉴定更多来自细胞-细胞连接的底物。在拟议的研究中，我们开发了体外CHO细胞聚集和解离试验来评估 细胞-细胞连接中的PS1/γ-分泌酶。我们已经发现，过量生产Nectin-1有利于聚集，反过来，降低细胞解离的能力。抑制这些细胞中PS1/伽马-分泌酶的活性可以减少约75%的解离，同时稳定与肌动蛋白细胞骨架结合的Nextin-1。同样，PS1/γ-分泌酶功能可能在细胞-细胞黏附的变化中调节其其他底物的周转，如 已经报告了E-钙粘素。我们假设，PS1或其底物(APP)的病理性突变可能会扰乱这些蛋白质周转的微妙平衡，其结果是改变Aβ的生成。为了跟进我们的初步结果，并扩展我们关于PS1/伽马分泌酶在细胞-细胞连接和Abeta产生中的作用的研究，我们建议解决两个主要的特定目标。首先，我们将评估PS1/伽马分泌酶活性在细胞-细胞连接形成和调节细胞-细胞连接蛋白功能中的作用(例如，通过使用已知的FAD和显性负突变和伽马分泌酶抑制剂)，然后评估对其底物加工和亚细胞定位的潜在影响，以及对体外细胞-细胞聚集/解离和原代神经元培养中突触形成的影响。 其次，我们将调控细胞与细胞的黏附，研究PS1/γ-分泌酶复合体的形成、定位和底物蛋白分解的相应变化，特别是对APP加工和Abeta产生的潜在影响。这项应用的首要目标是通过了解PS1/伽马分泌酶在细胞-细胞连接形成中的作用及其与Abeta产生的关系来确定PS基因FAD突变导致AD发病的致病机制。

PSl/gamma-secretase is a large protease complex mainly comprised of membrane proteins responsible for intramembranous cleavage of its substrates. PS1/gamma-secretase was originally shown to cleave the amyloid precursor protein (APP) and Notch, releasing the Abeta peptide and the Notch intracellular domain, respectively. We and others have recently identified additional substrates undergoing PS1-dependent proteolytic processing. Although these substrates perform a variety of different functions, we will explore the potential relationship of the roles played by the PS1/gamma-secretase complex specifically in Abeta generation and cell-cell junctions. We have recently identified a novel substrate for PS1/gamma-secretase activity, nectin-1, an adherens junction protein involved in synapse formation, and we are currently characterizing additional substrates from cell-cell junctions. For the proposed studies, we have developed in vitro CHO cell aggregation and dissociation assays to assess the role of PS1/gamma-secretase in cell-cell junctions. We have found that overproduction of nectin-1 favors aggregation and, conversely, lowers the ability of the cells to dissociate. Inhibition of PS1/gamma-secretase activity in these cells reduces dissociation by approximately 75%, while stabilizing nectin-1 bound to the actin cytoskeleton. Similarly, PS1/gamma-secretase function is likely to regulate the turnover of its other substrates during changes in cell-cell adhesion, as already reported for E-cadherin. We hypothesize that pathological mutations in PS1 or its substrates (APP) may perturb a delicate balance in the turnover of these proteins a consequence of which is altered Abeta generation. To follow up our preliminary results and extend our studies regarding the role of PS1/gamma-secretase in cell-cell junctions and Abeta generation, we propose to address two main specific aims. First, we will assess the role of PS1/gamma-secretase activity in cell-cell junction formation and in modulating the function of cell-cell junction proteins by modulating PS1/gamma-secretase activity (e.g. by using known FAD and dominant negative mutations and gamma-secretase inhibitors) and then evaluating potential effects on the processing and subcellular localization of its substrates as well as effects on cell-cell aggregation/dissociation in vitro and synapse formation in primary neuronal cultures. Second, we will modulate cell-cell adhesion and study corresponding changes in PS1/gamma-secretase complex formation, localization and proteolysis of its substrates, paying particular attention to potential effects on APP processing and Abeta generation. The overarching goal of this application is to define the pathogenic mechanism by which FAD mutations in the PS genes lead to the onset of AD by understanding the role of PS1/gamma-secretase in the formation of cell-cell junctions and its relationship to Abeta production.