Role of Rer1 in the regulation of gamma-secretase trafficking and activity

描述(申请人提供)：包含早老素(PS1或PS2)、尼卡司他素(NCT)、APH-1和PEN-2的伽马分泌酶复合体，催化β-淀粉样前体蛋白在膜内的蛋白分解，产生淀粉样β多肽(A2)，阿尔茨海默病(AD)的关键致病因子。有趣的是，编码PS1或PS2基因的致病突变已经在早发性家族性AD病例中被发现，所有这些PS突变导致更多淀粉样变性Abeta42肽的产生增加的机制未知(S)。由于Abeta(尤其是Abeta42)的积聚被认为会导致神经元功能障碍和死亡，因此调节3-分泌酶活性可能是治疗AD的一个有吸引力的策略。虽然我们对伽马分泌酶复合体的组装已有了很大的了解，但目前还不清楚伽马分泌酶在细胞内的转运是如何被调控的。由于不同亚细胞室的局部环境可能导致不同的Abeta物种的产生，阐明伽玛分泌酶运输的调控机制将对开发新的AD治疗策略具有重要意义。γ-分泌酶复合体组装在内质网(ER)中，但组装的复合体需要运输到分泌途径的晚期隔间，以遇到和处理其底物。正常情况下，绝大多数的伽马分泌酶保留在内质网中，只有一小部分存在于内质网的晚期。然而，当我们稳定地同时过表达APH-1、NCT、PS1和PEN-2时，超积累的伽马分泌酶复合体主要定位于质膜和早期内吞体内，这表明伽玛分泌酶ER保持所需的未知因子(S)在该复合体过表达后变得饱和，使酶泄漏到晚期隔室。我们最近确定Rer1p的人类同源物是一种PEN-2相互作用蛋白，Rer1p参与酵母中选定蛋白质的ER检索。根据(1)已知的酵母膜蛋白内质网回收功能，(2)它与伽马分泌酶复合体成分的相互作用，以及(3)我们的初步数据表明，质膜上成熟的NCT水平因Rer1过表达而降低，而通过Rer1表达下调而增加，我们假设Rer1是伽玛分泌酶内质网回收的限制因素，并建议表征其在伽玛分泌酶定位和活性调节中的作用(特定目标1)。在第二个目标中，我们建议检验一个假设，即家族性AD连锁PS1突变导致更多的伽马分泌酶复合体保留在内质网中，该隔室被证明优先产生Abeta42。我们将确定与疾病相关的PS1突变体对伽马分泌酶在细胞内定位的影响，以及它们与Abeta42产生的相关性。此外，我们将研究Rer1在(1)增加与疾病相关的PS1突变体的内质网保留率和(2)通过突变体增加Abeta42代中的作用。我们期望我们的研究结果将为治疗干预提供新的靶点，并促进我们对伽马分泌酶的整体生物学的理解。

DESCRIPTION (provided by applicant): Gamma-secretase complex containing presenilins (PS1 or PS2), nicastrin (NCT), APH-1 and PEN-2, catalyzes the intramembranous proteolysis of beta-amyloid precursor protein to generate amyloid beta peptides (A2), the key pathogenic player in Alzheimer's disease (AD). Interestingly, causative mutations in genes encoding PS1 or PS2 have been identified in early-onset familial AD cases and all of these PS mutations lead to increased production of the more amyloidogenic Abeta42 peptides by unknown mechanism(s). Since accumulation of Abeta (especially Abeta42) is believed to cause neuronal dysfunction and death, modulation of 3-secretase activity could be an attractive therapeutic strategy for AD. Although significant advances have been made to our understanding of the assembly of gamma-secretase complex, it is not clear how intracellular trafficking of gamma-secretase is regulated. Since the local environment in different subcellular compartments may contribute to the generation of different Abeta species, elucidating the regulatory mechanism of gamma-secretase trafficking will be valuable for the development of novel therapeutic strategies for AD. Gamma-secretase complex is assembled in the endoplasmic reticulum (ER), but the assembled complex needs to be transported to late compartments of the secretory pathway to encounter and process its substrates. Normally, the vast majority of gamma-secretase is retained in the ER and only a small fraction is present in the late compartments. However, when we stably overexpressed APH-1, NCT, PS1 and PEN-2 together, the hyperaccumulated gamma-secretase complex was predominantly localized on the plasma membrane and in early endosomes, suggesting that unknown factor(s) required for the ER retention of gamma-secretase become saturated upon overexpression of the complex, allowing the enzyme to leak into the late compartments. We recently identified the human homologue of Rer1p, the protein involved in the ER retrieval of selected proteins in yeast, as a PEN-2 interacting protein. Based on (1) its known function of ER retrieval of membrane proteins in yeast, (2) its interaction with gamma-secretase complex components and (3) our preliminary data showing that the levels of mature NCT on the plasma membrane are decreased by Rer1 overexpression and increased by downregulation of Rer1 expression, we hypothesize that Rer1 is the limiting factor for the ER retrieval of gamma-secretase and propose to characterize its role in the regulation of gamma-secretase localization and activity (Specific Aim 1). In the second aim, we propose to test a hypothesis that familial AD-linked PS1 mutations cause more gamma-secretase complex retained in the ER, the compartment that was shown to preferentially generate Abeta42. We will determine the effects of the disease- linked PS1 mutants on the intracellular localization of gamma-secretase and their relevance with Abeta42 production. In addition, we will examine the role of Rer1 in the (1) increased ER retention of the disease-linked PS1 mutants and (2) increased Abeta42 generation by the mutants. We expect that results of our investigations will provide new targets for therapeutic interventions as well as advance our understanding of the overall biology of gamma-secretase.