TRANSMEMBRANE APP PROCESSING BY GAMMA-SECRETASE

β-淀粉样蛋白是淀粉样蛋白的主要成分 阿尔茨海默病(AD)中发现的斑块。……位置的变化 Abeta的C-末端与前体的切割定义了 淀粉样蛋白促进能力与Abeta的神经毒性潜能 形成了。所有的C-末端多肽位点，都被蛋白酶切割 被称为伽马分泌酶，埋藏在膜双层内。近期 研究结果已经确定了所有这些人的生理学特征 已知的家族性、遗传性阿尔茨海默病：生产过剩， 更多的淀粉样变性和毒性形式的Abeta。这一观察结果强调了 γ-分泌酶在阿尔茨海默病病理生理学中的重要性。 我们已经建立了一个研究稀有的伽马分泌酶的模型系统。 培养细胞中过度表达底物的切割事件 蛋白水解酶是APP(C/1-102)C-99蛋白水解区的类似物。 该系统允许对底物和两种产品进行定量 蛋白水解性切割。蛋白水解性已定位于 ER或早期高尔基人，与独立的证据线一致 其他实验室。裂解部位对应于 非常长的Abeta(最高可达Abeta/45)。初步结果显示 伽马分泌酶活性作用于膜结合的底物 留下至少一种膜相关产物。这一结果表明 史无前例的伽马分泌酶裂解氨基酸区的模型 通常被埋在脂质双层内，而不分离 薄膜。 我们建议，首先，确认前体的膜缔合 和产品。我们将通过以下几个方面来描述该协会的性质 实验方法，以了解的取向 底物在蛋白质分解过程中。我们还将检查膜 γ-分泌酶蛋白水解酶活性的关联性要求 以及在一定条件下细胞内Abeta是否是膜相关的 在那里形成但不会从细胞中释放出来。我们还将描述 过量生产C/t的细胞分泌的Abeta的C-末端序列- 102. 伽马分泌酶活性确切环境的特征 将提供对可能类型的生理性或 导致蛋白分解活性改变的细胞结构变化或 专一性。更清晰地定义需求和蜂窝位置 这种酶的活性将推动我们长期的努力，以识别和 提纯γ-分泌酶--抑制白血病的重要治疗靶点 与AD相关的神经变性。

Beta-amyloid peptide (Abeta or BetaA4) is the major component in amyloid plaques found in Alzheimer's Disease (AD). Variation in the position of cleavage at the C-terminal end of Abeta from the precursor define the amyloid promoting ability and the neurotoxic potential of the Abeta formed. All of the C-terminal peptide sites, cleaved by the protease termed gamma-secretase, are buried within the membrane bilayer. Recent findings have identified a unifying feature in the physiology of all the known familial, hereditary forms of AD: over-production of the longer, more amyloidogenic and toxic forms of Abeta. This observation underscores the importance of gamma-secretase in the pathophysiology of AD. We have developed a model system for studying the rare gamma-secretase cleavage event in cultured cells that over-express the substrate for the protease, an analogue of the C-99 proteolytic fragment of APP (C/1-102). This system allow quantitation of the substrate and both products of proteolytic cleavage. The proteolytic activity has been localized to the ER or early golgi, in agreement with independent lines of evidence from other laboratories. The cleavage site corresponds to the formation of very long froms of Abeta (up to Abeta/45). Preliminary results indicate that the gamma-secretase activity acts upon a membrane-bound substrate leaving at least one membrane associated product. This result suggests an unprecedented model in which gamma-secretase cleaves an amino acid domain that is ordinarily buried within the lipid bilayer without separating the membrane. We propose, first, to confirm the membrane association of the precursor and product. We will characterize the nature of the association by several experimental approaches in order to understand the orientation of substrate during proteolytic cleavage. We will also examine the membrane association requirements of the gamma-secretase proteolytic activity(ies) and whether intracellular Abeta is membrane associated under conditions where it is formed but not released from cells. We will also characterize the C-terminal sequence of the Abeta secreted by cells over-producing C/t- 102. Characterization of the exact milieu in which gamma-secretase is active will provide insight into the possible types of physiological or structural changes in cells that lead to altered proteolytic activity or specificity. Clearer definition of the requirements and cellular location of the enzyme activity will advance our long-term effort to identify and purify gamma-secretase, an important therapeutic target for the arrest of AD-associated neurodegeneration.