PURIFICATION AND RECONSTITUTION OF ACTIVE GAMMA SECRETASE COMPLEX

活性伽玛分泌酶复合物的纯化和重构

• 批准号: 7483170
• 负责人: DENNIS J SELKOE
• 金额: $ 42.79万
• 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/7483170
• 关键词: Active Sites; Address; Affinity; Alzheimer' s Disease; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Aspartate; Aspartic Endopeptidases; Binding; Biochemical; Biochemical Genetics; Biological Assay; Biology; C83; Caenorhabditis elegans; Calcium; Cells; Chemicals; Class; Cleaved cell; Complex; Cytoplasmic Protein; Detergents; Development; Disease; Docking; Endopeptidases; Environment; Enzymes; Genetic; Goals; Guanosine Triphosphate; Health; In Vitro; Integral Membrane Protein; Ions; Kinetics; Knowledge; Laboratories; Light; MTCH1 gene; Mammalian Cell; Mass Spectrum Analysis; Mediator of activation protein; Membrane Proteins; Metabolic; PVRL1; Peptide Hydrolases; Phospholipids; Property; Proteins; Proteolysis; Protocols documentation; Range; Rate; Research; Signal Pathway; Therapeutic; Time; Transmembrane Domain; Vesicle; Work; Yeasts; base; beta-site APP cleaving enzyme 1; calsenilin; cofactor; design; dimer; gamma secretase; human APH-1 protein; human PEN-2 protein; inhibitor/antagonist; interest; member; nicastrin protein; notch protein; novel; presenilin; reconstitution; scale up; secretase; size; stoichiometry

Gamma-Secretase is an unusual enzyme that catalyzes the intramembrane proteolysis of numerous type I integral membrane proteins. These include the amyloid-beta protein precursor (APP), which releases the amyloid-beta peptide (Abeta) implicated in Alzheimer's disease (AD). For this reason, gamma-secretase is a prime target for the development of therapeutic and preventative agents for AD. Pharmacological, genetic, and biochemical evidence generated under this project strongly supports the hypothesis that the polytopic membrane protein, presenilin (PS), is the catalytic component of a larger multi-protein gamma-secretase complex. PS is normally cleaved into two fragments that remain associated. These heterodimers are metabolically stable, and their formation is tightly regulated by limiting cellular factors. During the current project period, we discovered that both PS endoproteolysis and gamma-secretase activity require two conserved transmembrane aspartates, consistent with our provocative hypothesis that presenilin undergoes autoproteolysis to become the catalytic component of a novel aspartyl protease. Moreover, using affinity isolation on a designed inhibitor, we showed that the PS-associated protein, nicastrin, is another member of the complex. Recently, our laboratory has found that the co-expression of PS1, nicastrin, and two proteins identified in C. elegans, aph-1 and pen-2, results in increased PS heterodimer formation, full nicastrin maturation and enhanced gamma-secretase activity. All 4 proteins co-immunoprecipitate and bind to a gamma-secretase affinity matrix. Taken together, these new finding suggest that PS, nicastrin, aph-1, and pen-2 assemble as a complex that leads to the formation of active gamma-secretase. In light of these discoveries, we now propose to purify, fully characterize and then reconstitute active gamma-secretase. Specifically, we will: 1) scale up and refine the multi-step purification protocols for gamma-secretase established by our labs in order to unequivocally confirm the presence of these 4 key components and search for any additional protein factors by mass spectrometry; 2) systematically explore phospholipid, detergent, ionic, energy, pH and other requirements to optimize our established in vitro cleavage assay (which can generate Abeta, AICD and NICD) and characterize the kinetic properties of the protease; and 3) using this new knowledge, carry out the step-wise addition of recombinantly expressed PS, nicastrin, aph-1, and pen-2 in a pure detergent/phospholipid environment to reconstitute proteolysis. The purification, detailed characterization, and reconstitution of gamma-secretase will aid the understanding of this essential protease in health and disease and the search for effective and safe AD therapeutics. Moreover, this work will advance our fundamental knowledge of a unique new class of intramembrane-cleaving proteases.

γ-分泌酶是一种不寻常的酶，可催化许多I型积分膜蛋白的膜内蛋白水解。其中包括淀粉样蛋白蛋白前体（APP），该蛋白蛋白（APP）释放了与阿尔茨海默氏病（AD）有关的淀粉样蛋白β肽（ABETA）。因此，γ-分泌酶是开发AD治疗和预防剂的主要目标。在该项目下产生的药理，遗传和生化证据强烈支持以下假设：多重膜蛋白Presenilin（PS）是较大的多蛋白质伽玛 - 分泌酶配合物的催化成分。 PS通常被裂解成两个保持相关的片段。这些异二聚体在代谢上是稳定的，它们的形成受到细胞因子的严格调节。在当前的项目期间，我们发现PS内蛋白水解和伽马分泌酶均可 活性需要两个保守的跨膜天冬氨酸，这与我们的挑衅性假设一致，即在挑衅性的假设中，培养基蛋白经历了自相度为新的天冬氨酸蛋白酶的催化成分。此外，在设计的抑制剂上使用亲和力隔离，我们表明与PS相关的蛋白质尼卡斯特林是复合物的另一个成员。最近，我们的实验室发现，在秀丽隐杆线虫，APH-1和PEN-2中鉴定的PS1，Nicastrin和两种蛋白的共表达导致PS异二聚体形成增加，全尼里卡斯特蛋白 成熟和增强的γ-分泌酶活性。所有4种蛋白质共免疫沉淀并与γ-分泌酶亲和力基质结合。综上所述，这些新发现表明，PS，Nicastrin，APH-1和PEN-2作为一种复合物，导致活性γ-分泌酶的形成。鉴于这些发现，我们现在建议净化，充分表征并重建活跃的γ-分泌酶。具体而言，我们将：1）我们实验室建立的γ-分泌酶的多步纯化方案进行扩展和完善，以明确确认这4个关键组件的存在，并通过质谱法搜索任何其他蛋白质因子； 2）系统地探索磷脂，清洁剂，离子，能量，pH和其他要求，以优化我们已建立的体外切割测定法（可以生成Abeta，AICD和NICD），并表征蛋白酶的动力学特性； 3）使用这些新知识，在纯洗涤剂/磷脂环境中逐步添加重组表达的PS，Nicastrin，APH-1和PEN-2，以重建蛋白水解。 γ-分泌酶的纯化，详细的表征和重构将有助于理解健康和疾病中这种基本蛋白酶，并寻求有效且安全的AD疗法。此外，这项工作将促进我们对独特的新膜内切割蛋白酶的基本知识。