RAB GTPASES AND TRAFFICKING OF BETA AMYLOID PROTEINS

淀粉样β-肽(Abeta)是脑内淀粉样斑块的主要成分。 阿尔茨海默氏症。Abeta是通过细胞内的蛋白分解形成的 膜锚定糖蛋白β-淀粉样蛋白的加工 前体蛋白(APP)。细胞可以产生不同形式的Abeta (例如，Abeta40和Abeta42)，并且Abeta42具有最大的倾向 形成不溶的沉淀物。我们的一般假设是相对的 细胞产生和释放的Abeta42和Abeta40的量是 至少部分决定于蛋白质贩运的水平。我们的 长期目标是定义符合以下条件的特定交通事件 对(1)APP C-末端的淀粉样蛋白形成过程至关重要 被胞内蛋白酶(S)称为伽马分泌酶的片段，和(2) 随后从细胞中释放出不同形式的Abeta。至 为了实现这一目标，分子和病毒转基因策略将是 用于在培养细胞中表达显性-负性Rab突变体。自.以来 不同的Rab GTP酶在囊泡转运中的作用 在胞外和胞外的特定供体和受体隔间之间 内吞途径，这一策略将允许我们选择性地破坏 可能作为交付前体的基础的离散的贩运步骤 到含有γ-分泌酶活性的细胞器的多肽和 将最终的Abeta产物输送到细胞外环境。 为了便于识别神经元中可能不同的步骤 与未分化细胞相比，研究将在两种情况下进行 NT2N神经元和人胚胎肾细胞(HEK293)。通过表达 不同的Rab突变体，其APP的形式发生了变化，其中包含突变 在家族性阿尔茨海默病中发现，应该可以确定 具有特别相关性的具体贩运步骤 从这些改变的前体中增加Abeta的产生。这些 研究将提供有关亚细胞的新信息 产生的伽马分泌酶活动的分区 不同形式的Abeta，并帮助定义这些 产品从细胞中释放出来。这一信息可能有助于 制定针对相关疾病的治疗策略 蛋白水解酶，特别是伽马分泌酶 产生致病的Abeta42亚型。

Amyloid beta-peptide (Abeta) is a major component of amyloid plaques in Alzheimer's disease. Abeta is formed through intracellular proteolytic processing of a membrane-anchored glycoprotein termed beta-amyloid precursor protein (APP). Cells can produce alternate forms of Abeta (e.g., Abeta40 and Abeta42), and Abeta42 has the greatest tendency to form insoluble deposits. Our general hypothesis is that the relative amounts of Abeta42 and Abeta40 generated and released by the cell are determined, at least in part, at the level of protein trafficking. Our long range goal is to define the specific transport events that are critical for (1) the amyloidogenic processing of APP C-terminal fragments by intracellular protease(s) termed gamma-secretase, and (2) the subsequent release of different forms of Abeta from the cell. To pursue this goal, molecular and viral transfection strategies will be used to express dominant-negative Rab mutants in cultured cells. Since different Rab GTPases function as mediators of vesicular transport between specific donor and acceptor compartments in the exocytic and endocytic pathways, this strategy will allow us to selectively disrupt discrete trafficking steps that may underlie the delivery of precursor peptides to organelles containing gamma-secretase activity and the delivery of the final Abeta products to the extracellular environment. To facilitate the identification of steps that may vary in neurons versus non-differentiated cells, studies will be carried out in both NT2N neurons and human embryonal kidney cells (HEK293). By expressing various Rab mutants with altered forms of APP that harbor mutations found in familial Alzheimer's disease, it should be possible to identify specific trafficking steps that have particular relevance for the increased production of Abeta from these altered precursors. These studies will provide new information about the subcellular compartmentalization of the gamma-secretase activities that give rise to different forms of Abeta, and help define the routes whereby these products are released from the cell. This information may facilitate the development of therapeutic strategies targeted at the relevant proteases, particularly the gamma-secretase responsible for the production of the pathogenic Abeta42 isoform.