STRUCTURE, FUNCTION AND REGULATION OF GAMMA-SECRETASE

伽马分泌酶的结构、功能和调节

• 批准号: 8738548
• 负责人: OKSANA BEREZOVSKA
• 金额: $ 69.62万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8738548
• 关键词: Affinity Labels; Alzheimer' s Disease; Amyloid; Amyloid beta-Protein; Aspartic Endopeptidases; Biochemistry; Biological Models; Biology; Brain; Cell membrane; Cells; Cleaved cell; Complex; Critiques; Crystallography; Cut protein; Data; Environment; Enzymes; Grant; HLA-A2 Antigen; Head; Human; In Vitro; Individual; Instruction; Laboratories; Life; Lipids; Mammalian Cell; Mediating; Membrane; Membrane Lipids; Modeling; Mus; N-Cadherin; Neurons; Outcome; PVRL1; Pathway interactions; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Play; Process; Production; Protein Structure Initiative; Proteins; Proteolysis; Reaction; Regulation; Research; Resolution; Role; Seminal; Side; Signal Pathway; Site; Stretching; Structural Biologist; Structure; Structure-Activity Relationship; Testing; Universities; Water; Work; amyloid precursor protein processing; base; beta secretase; cell type; cofactor; design; experience; gamma secretase; human PEN-2 protein; in vivo; inhibitor/antagonist; member; nicastrin protein; notch protein; presenilin; protein B; protein complex; prototype; reconstitution; research study; secretase; site-1 protease; three dimensional structure

The discovery of presenilin (PS) as the first intramembrane aspartyl protease and the catalytic center of gamma-secretase occurred in Project 1. Since then, >100 substrates have been identified. PS/gamma-secretase mediates critical signaling pathways necessary for life in all metazoans, and its cleavage of APP releases the amyloid B-protein that accumulates in all patients with AD. After identifying PS as a protease, Project 1 has continued to contribute actively to PS biology. We proposed - and provided the first evidence - that holoPS undergoes autocatalytic endoproteolysis to generate the active heterodimer, first reconstituted PS and its 3 cofactors in mammalian cells, purified the protease to homogeneity, obtained the first 3D structure of the ycomplex by EM, conducted SILAC screens to identify several new substrates, and designed many gamma-secretase inhibitors, some of which are potent and much more APP-selective than compounds tried in humans. The Project is now revised to respond to all of the SEP's helpful critiques. We propose to study 4 related topics in the biochemistry of gamma-secretase. 1: A new cell biological model of secretase processing We have discovered that contrary to current concepts, alpha- (ADAM 10), beta- and gamma-secretases exist in part in a large protein complex that can mediate efficient sequential processing of substrates. Our extensive supporting data include robust co-IP of endogenous a- and gamma-secretases from wt brain and the sequential alpha/gamma processing of an APP substrate. We propose to fully confirm this new model of regulated intramembrane proteolysis and ask if it generalizes to another membrane protease pair: S1P/S2P. 2: The complex regulation of gamma-secretase by membrane lipids. We will extend our recent evidence that certain head groups and fatty acyl side chains of membrane lipids potently up- and down-regulate gamma-cleavages, including the key A(i42/40 ratio. We'll seek to validate robust in vitro effects of certain lipids by manipulating their cognate biosynthetic and catabolic enzymes in vivo. 3: Toward greater structural resolution of the gamma-secretase complex Working with leading structural biologists, we will pursue the technically challenging but essential quest for the structure of gamma-secretase via: a) further cryo-EM analyses of 2D crystals; b) 3D x-ray crystallography of individual gamma-components (PS, Net, Pen-2); and c) attempted 3D x-ray crystallography of the purified holo-enzyme. 4: Refining potent and selective Notch-sparing gamma-inhibitors and defining their mechanism. Building on more than 1,600 compounds we've synthesized, we will develop SARs for inhibiting APP vs. Notch, and for the most selective compounds, assess cleavage of other gamma-substrates and test them in mice. These aims build on our experience to tackle some of the thorniest problems in gamma-secretase biology.

早老素（PS）作为第一个膜内乙酰化蛋白酶和γ-分泌酶催化中心的发现 发生在项目1。从那时起，已经确定了>100种底物。PS/γ-分泌酶介导 所有后生动物生命所必需的关键信号通路，其裂解APP释放淀粉样蛋白 B蛋白在所有AD患者中积累。在确定PS为蛋白酶后，项目1 继续为PS生物学做出积极贡献。我们提出并提供了第一个证据， 经历自催化内蛋白水解产生活性异二聚体，首先重建PS及其3 在哺乳动物细胞中的辅因子，纯化的蛋白酶的同质性，获得了第一个三维结构的y复合物 利用EM技术，进行SILAC筛选，鉴定出几种新的底物，并设计了多种γ-分泌酶 抑制剂，其中一些是有效的，更APP选择性比化合物尝试在 人类该项目现已修订，以回应所有的SEP的有益的批评。我们建议 研究γ-分泌酶生物化学中的4个相关课题。1：一种新的分泌酶细胞生物学模型 我们已经发现，与目前的概念相反，存在α-（ADAM 10），β-和γ-分泌酶， 部分地存在于大蛋白复合物中，所述大蛋白复合物可以介导底物的有效顺序加工。我们 广泛的支持数据包括来自野生型脑的内源性α-和γ-分泌酶的稳健的co-IP和来自野生型脑的内源性α-和γ-分泌酶的稳健的co-IP。 APP基底的连续α/γ处理。我们建议充分确认这种新的受监管模式， 膜内蛋白水解，并询问它是否推广到另一种膜蛋白酶对：S1 P/S2 P。2：The 膜脂对γ-分泌酶的复杂调节。我们将扩展我们最近的证据， 膜脂质的基团和脂肪酰基侧链有效地上调和下调γ-裂解，包括 键A（i42/40比率。我们将试图通过操纵某些脂质的体外作用来验证它们的强大作用。 体内同源生物合成和分解代谢酶。3：γ-分泌酶的更高结构分辨率 与领先的结构生物学家合作，我们将追求技术上具有挑战性，但 通过以下方式对γ-分泌酶的结构进行基本探索：a）对2D晶体进行进一步的冷冻EM分析; B）3D X射线 单个γ-组分（PS、Net、Pen-2）的晶体学;和c） 纯化的全酶4：提炼有效的和选择性的缺口保留γ抑制剂，并确定其 机制在我们合成的1,600多种化合物的基础上，我们将开发SAR， APP vs. Notch，对于最具选择性的化合物，评估其他γ-底物的裂解并对其进行测试 对小鼠这些目标建立在我们解决γ分泌酶生物学中一些最棘手问题的经验基础上。