GAMMA SECRETASE ACTIVITY N COORDINATED CELL-CELL INTERACTIONS

伽玛分泌酶活性与协调的细胞间相互作用

• 批准号: 7483175
• 负责人: DORA M KOVACS
• 金额: $ 47.27万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7483175
• 关键词: Abeta synthesis; Actins; Address; Adherens Junction; Amyloid; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Attention; Binding; Biological Assay; Brain; C-terminal; CD44 gene; Cell Adhesion; Cell Aggregation; Cell Communication; Cell physiology; Cell surface; Cell-Cell Adhesion; Cells; Chinese Hamster Ovary Cell; Cleaved cell; Complex; Cytoskeleton; Disruption; Dissociation; Dominant-Negative Mutation; E-Cadherin; Endopeptidases; Equilibrium; Event; Gene-Modified; Generations; Genes; Genetic Polymorphism; Goals; In Vitro; Intercellular Junctions; Lead; Maintenance; Mediating; Membrane Proteins; Multienzyme Complexes; Mus; Mutation; N-Cadherin; Nerve Degeneration; Neurites; Neurons; PVRL1; Pathogenesis; Patients; Peptide Hydrolases; Peptides; Play; Presenile Alzheimer Dementia; Process; Production; Protein C; Proteins; Proteolysis; Proteolytic Processing; Range; Rate; Reporting; Role; Role playing therapy; Signal Transduction; Sodium Channel; Testing; Transmembrane Domain; amyloid precursor protein processing; association cortex; beta-site APP cleaving enzyme 1; computerized data processing; familial Alzheimer disease; follow-up; gamma secretase; inhibitor/antagonist; migration; nectin; normal aging; notch protein; novel; novel therapeutics; peptide A; protein degradation; response; secretase; synaptogenesis; syndecan 3; therapeutic target; trafficking; voltage

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.

PS1/γ-分泌酶是主要由负责其底物的膜内切割的膜蛋白组成的大型蛋白酶复合物。PS1/γ-分泌酶最初显示切割淀粉样前体蛋白（APP）和Notch，分别释放Abeta肽和Notch细胞内结构域。我们和其他人最近已经确定了额外的基板进行PS1依赖的蛋白水解加工。虽然这些基板执行各种不同的功能，我们将探讨的PS1/γ-分泌酶复合物所发挥的作用，特别是在Abeta生成和细胞-细胞连接的潜在关系。我们最近发现了一种新的基板PS1/γ-分泌酶活性，nectin-1，参与突触形成的粘附连接蛋白，我们目前正在表征额外的基板从细胞-细胞连接。对于拟议的研究，我们开发了体外CHO细胞聚集和解离测定，以评估 细胞-细胞连接中的PS1/γ-分泌酶。我们已经发现，nectin-1的过量产生有利于聚集，相反，降低了细胞解离的能力。在这些细胞中抑制PS1/γ-分泌酶活性可使解离减少约75%，同时稳定与肌动蛋白细胞骨架结合的连接蛋白-1。类似地，PS1/γ-分泌酶功能可能在细胞-细胞粘附变化期间调节其其他底物的周转，如 已经报告了E-钙粘蛋白我们假设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Abeta的产生。为了跟进我们的初步结果，并扩大我们的研究PS1/γ-分泌酶在细胞连接和Abeta生成的作用，我们建议解决两个主要的具体目标。第一、我们将评估PS1/γ-分泌酶活性在细胞-细胞连接形成中的作用，以及通过调节PS1/γ-分泌酶活性来调节细胞-细胞连接蛋白的功能（例如，通过使用已知的FAD和显性负突变和γ-分泌酶抑制剂），然后评估对其底物的加工和亚细胞定位的潜在影响以及对细胞-细胞聚集的影响。体外解离和原代神经元培养物中的突触形成。 其次，我们将调节细胞间粘附，并研究PS1/γ-分泌酶复合物形成、定位及其底物蛋白水解的相应变化，特别关注对APP加工和Abeta产生的潜在影响。本申请的首要目标是通过了解PS1/γ-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，确定PS基因中FAD突变导致AD发作的致病机制。