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，可催化多种 I 型整合膜蛋白的膜内蛋白水解。其中包括 β 淀粉样蛋白前体 (APP)，它会释放与阿尔茨海默病 (AD) 相关的 β 淀粉样肽 (Abeta)。因此，γ-分泌酶是开发 AD 治疗和预防药物的主要靶点。该项目产生的药理学、遗传和生化证据强烈支持这样的假设：多胞膜蛋白早老素（PS）是更大的多蛋白γ-分泌酶复合物的催化成分。 PS 通常被切割成两个保持关联的片段。这些异二聚体代谢稳定，它们的形成受到限制性细胞因素的严格调控。在当前项目期间，我们发现 PS 内切蛋白水解酶和 γ-分泌酶 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，即早老素经历自身蛋白水解，成为新型天冬氨酰蛋白酶的催化成分。此外，通过对设计的抑制剂进行亲和分离，我们发现 PS 相关蛋白尼卡斯特林是该复合物的另一个成员。最近，我们的实验室发现PS1、尼卡斯特林和在线虫中鉴定的两种蛋白质aph-1和pen-2的共表达，导致PS异二聚体形成增加，全尼卡斯特林 成熟和增强的γ-分泌酶活性。所有 4 种蛋白质均免疫共沉淀并与 γ-分泌酶亲和基质结合。总而言之，这些新发现表明 PS、尼卡斯特林、aph-1 和 pen-2 组装成复合物，导致活性伽马分泌酶的形成。鉴于这些发现，我们现在建议纯化、充分表征并重建活性伽马分泌酶。具体来说，我们将：1）扩大和完善我们实验室建立的γ-分泌酶多步纯化方案，以便明确确认这 4 个关键成分的存在，并通过质谱法寻找任何其他蛋白质因子； 2）系统地探索磷脂、去垢剂、离子、能量、pH等要求，以优化我们建立的体外裂解实验（可生成Abeta、AICD和NICD）并表征蛋白酶的动力学特性； 3) 利用这一新知识，在纯洗涤剂/磷脂环境中逐步添加重组表达的 PS、尼卡斯特林、aph-1 和 pen-2，以重建蛋白水解。 γ-分泌酶的纯化、详细表征和重建将有助于了解这种健康和疾病中必需的蛋白酶，并有助于寻找有效和安全的 AD 疗法。此外，这项工作将增进我们对独特的新一类膜内切割蛋白酶的基础知识。