PURIFICATION AND RECONSTITUTION OF ACTIVE GAMMA SECRETASE COMPLEX

γ-分泌酶是一种不常见的酶，它催化许多I型完整膜蛋白的膜内蛋白分解。其中包括淀粉样β蛋白前体(APP)，它释放与阿尔茨海默病(AD)有关的淀粉样β蛋白(Abeta)。因此，伽玛分泌酶是开发AD治疗和预防药物的主要靶点。在该项目下产生的药理学、遗传学和生化证据有力地支持了这样的假设，即多表膜蛋白早老素(PS)是更大的多蛋白伽玛分泌酶复合体的催化成分。PS通常被裂解成两个保持关联的片段。这些异源二聚体在代谢上是稳定的，它们的形成受到限制细胞因子的严格调控。在本项目期间，我们发现PS内切和伽马-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相一致，即早老素经历了自蛋白分解，成为一种新型天冬氨酸蛋白酶的催化成分。此外，通过对设计的抑制剂进行亲和分离，我们表明PS相关蛋白NICASTIN是该复合体的另一个成员。最近，我们的实验室发现，在线虫中，ps1、Nicastin和两种已鉴定的蛋白质aph-1和pen-2的共同表达导致PS异源二聚体的形成增加，即完整的Nicastin。 成熟和增强的伽马分泌酶活性。所有4种蛋白质都能免疫共沉淀并结合到一个伽马分泌酶亲和基质上。综上所述，这些新发现表明PS、尼古丁、APH-1和PEN-2作为一个复合体组装在一起，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、充分鉴定并重建活性伽马分泌酶。具体地说，我们将：1)扩大和完善我们实验室建立的伽马分泌酶的多步纯化方案，以明确地确认这4个关键成分的存在，并通过质谱学寻找任何额外的蛋白质因素；2)系统地探索磷脂、洗涤剂、离子、能量、pH和其他要求，以优化我们建立的体外切割试验(可以产生Abeta、AICD和NICD)并表征该酶的动力学性质； 3)利用这一新知识，在纯净的洗涤剂/磷脂环境中，逐步将重组表达的PS、NICASTIN、APH-1和PEN-2相加，以重建蛋白降解。对γ-分泌酶的纯化、详细鉴定和重组将有助于了解这一在健康和疾病中必不可少的酶，并有助于寻找有效和安全的AD治疗方法。此外，这项工作将增进我们对一类独特的膜内裂解酶的基础知识。

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.