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

描述（由申请人提供）：含有早老素（PS1或PS2）、nicastrin （NCT）、APH-1和PEN-2的γ -分泌酶复合物，催化β -淀粉样蛋白前体蛋白的膜内蛋白水解生成淀粉样蛋白β肽（A2），这是阿尔茨海默病（AD）的关键致病因子。有趣的是，在早发性家族性AD病例中发现了编码PS1或PS2基因的致病突变，所有这些PS突变都以未知的机制导致淀粉样蛋白Abeta42肽的产生增加。由于Abeta（尤其是Abeta42）的积累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的一种有吸引力的策略。尽管我们对γ -分泌酶复合物组装的理解取得了重大进展，但细胞内γ -分泌酶的运输是如何调节的尚不清楚。由于不同亚细胞区室的局部环境可能导致不同Abeta物种的产生，阐明γ -分泌酶运输的调控机制将对开发新的AD治疗策略有价值。γ -分泌酶复合物在内质网（ER）中组装，但组装的复合物需要被运输到分泌途径的后期区室，以遇到并处理其底物。正常情况下，绝大部分γ -分泌酶保留在内质网中，只有一小部分存在于后腔室。然而，当我们稳定地过表达APH-1、NCT、PS1和PEN-2时，高积累的γ -分泌酶复合物主要定位于质膜和早期内体，这表明γ -分泌酶的内质网保留所需的未知因子在复合物过表达时变得饱和，从而使酶渗漏到晚期区室。我们最近发现了与酵母菌中特定蛋白的内质网检索有关的蛋白Rer1p的人类同源物，作为一个PEN-2相互作用蛋白。基于(1)其已知的酵母膜蛋白ER检索功能，(2)其与γ -分泌酶复合物组分的相互作用，以及(3)我们的初步数据显示，质膜上成熟的NCT水平通过过表达Rer1而降低，并通过下调Rer1表达而增加。我们假设Rer1是γ -分泌酶在内质网中检索的限制因素，并提出表征其在调节γ -分泌酶定位和活性中的作用（Specific Aim 1）。在第二个目标中，我们提出验证一个假设，即家族性ad相关的PS1突变导致内质网中保留更多的γ -分泌酶复合物，内质网被证明优先产生Abeta42。我们将确定疾病相关的PS1突变体对γ -分泌酶细胞内定位的影响及其与Abeta42产生的相关性。此外，我们将研究Rer1在以下方面的作用：(1)疾病相关的PS1突变体增加内质网保留，(2)突变体增加Abeta42的产生。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γ分泌酶整体生物学的理解。

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.