TRANSMEMBRANE APP PROCESSING BY GAMMA-SECRETASE

β-淀粉样肽（Abeta或BetaA 4）是淀粉样蛋白的主要成分， 阿尔茨海默病（AD）中发现的斑块。的位置变化 在Abeta的C-末端从前体的裂解定义了 淀粉样蛋白促进能力和Abeta的神经毒性潜力 形成了所有的C-末端肽位点，被蛋白酶切割 称为γ-分泌酶，被埋在膜双层内。最近 研究结果已经确定了一个统一的功能，在生理学的所有 已知的家族性、遗传性AD形式：较长的， 淀粉样蛋白和毒性更强的A β这一观察强调， γ-分泌酶在AD病理生理学中的重要性。 我们开发了一个研究罕见的γ-分泌酶的模型系统 在培养的细胞中的裂解事件，所述细胞过表达所述底物， 蛋白酶，APP（C/1-102）的C-99蛋白水解片段的类似物。 该系统允许定量底物和底物的两种产物。 蛋白水解裂解。蛋白水解活性已定位于 ER或早期高尔基体，与来自 其他实验室。切割位点对应于 很长的Abeta形式（高达Abeta/45）。初步结果表明 γ-分泌酶的活性作用于膜结合底物 留下至少一种膜相关产物。这一结果表明， 前所未有的模型，其中γ-分泌酶切割氨基酸结构域 它通常埋在脂质双层内，而不分离脂质双层。 膜的 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协会的性质： 实验方法，以了解方向 蛋白水解裂解过程中的底物。我们还将检查细胞膜 γ-分泌酶蛋白水解活性的结合要求 以及在一定条件下细胞内Abeta是否与膜结合 在那里它形成但不从细胞中释放。我们还将描述 由过度产生C/t的细胞分泌的Abeta的C-末端序列， 102. γ-分泌酶活性的确切环境的表征 将提供对可能类型的生理或 导致蛋白水解活性改变的细胞结构变化，或 的特异性更明确地定义要求和蜂窝位置 酶的活性将促进我们的长期努力，以确定和 纯化γ-分泌酶，一个重要的治疗目标， AD相关神经变性。

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.