PURIFICATION AND RECONSTITUTION OF ACTIVE GAMMA SECRETASE COMPLEX

γ-分泌酶是一种不寻常的酶，催化许多I型整合膜蛋白的膜内蛋白水解。这些包括淀粉样β蛋白前体（APP），其释放与阿尔茨海默病（AD）有关的淀粉样β肽（Abeta）。因此，γ-分泌酶是开发AD治疗和预防剂的主要靶标。药理学，遗传学和生物化学的证据下产生的这个项目强烈支持的假设，多位膜蛋白，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。PS通常被切割成两个保持结合的片段。这些异二聚体是代谢稳定的，并且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PS内蛋白水解和γ-分泌酶 活性需要两个保守的跨膜磷酸盐，与我们的挑衅性假设一致，即早老素经历自蛋白水解成为一种新的乙酰基蛋白酶的催化组分。此外，使用设计的抑制剂上的亲和分离，我们表明，PS相关蛋白，nicastrin，是另一个成员的复杂。最近，我们实验室发现PS 1、nicastrin和两种在C.线虫，aph-1和pen-2，导致PS异二聚体形成增加，完全nicastrin 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-分泌酶亲和基质结合。综上所述，这些新发现表明PS、nicastrin、aph-1和pen-2组装成复合物，导致活性γ-分泌酶的形成。根据这些发现，我们现在建议纯化，充分表征，然后重建活性γ-分泌酶。具体而言，我们将：1）扩大和完善我们实验室建立的γ-分泌酶的多步纯化方案，以明确确认这4种关键组分的存在，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、去污剂、离子、能量、pH等要求，优化我们建立的体外切割实验（其可产生Abeta、AICD和NICD）并表征蛋白酶的动力学性质; 和3）使用该新知识，在纯去污剂/磷脂环境中逐步加入重组表达的PS、nicastrin、aph-1和pen-2以重建蛋白水解。γ-分泌酶的纯化、详细表征和重建将有助于了解这种在健康和疾病中必不可少的蛋白酶，并有助于寻找有效和安全的AD治疗方法。此外，这项工作将推进我们对一类独特的新型膜内切割蛋白酶的基本知识。

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.