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 -分泌酶是一种罕见的酶，催化许多I型整体膜蛋白的膜内蛋白水解。这些包括淀粉样蛋白前体（APP），它释放与阿尔茨海默病（AD）有关的淀粉样蛋白肽（Abeta）。因此，分泌酶是开发AD治疗和预防药物的主要目标。该项目产生的药理学、遗传学和生化证据有力地支持了多聚膜蛋白早老素（PS）是一个更大的多蛋白γ -分泌酶复合物的催化成分的假设。PS通常被分裂成两个相互关联的片段。这些异源二聚体在代谢上是稳定的，它们的形成受到限制性细胞因子的严格调控。在目前的项目期间，我们发现了PS内蛋白水解和γ -分泌酶