Regulation and Cell Biology of Beta-Secretase

描述（由申请人提供）：沉积在阿尔茨海默病（AD）患者大脑中的淀粉样蛋白由4kDa淀粉样蛋白肽或a - β组成。a- β是由β和γ分泌酶对淀粉样蛋白前体蛋白（APP）的顺序切割产生的。一种新型的1型整膜天冬氨酸蛋白酶BACE已被确定为β分泌酶，敲除小鼠模型表明该酶是小鼠大脑中产生A- β所必需的。β -分泌酶生成a- β是APP加工的次要途径，导致10%的APP加工为其分泌衍生物。由于BACE在体外是一种活性较差的酶，因此有人提出它在细胞内的活性是有限的。然而，在表达高水平APP的转染细胞中，a - β的产生增加了几个数量级，而BACE活性却没有相应的增加。为了解释这些不同的观察结果，PI提出了一个假设，即BACE的APP处理发生在一个特殊的细胞室中，其活动不受限制。相反，只有一小部分APP进入这个隔间，这是BACE对APP处理的限制。这一假设与PI实验室最近的发现一致，表明BACE可以作为高活性的高分子量复合物从豚鼠大脑中分离出来。为了进一步验证这一假设，将产生1个APP嵌合体，将APP靶向到专门的细胞区室，并对其加工过程进行研究。在目标2中，我们将评估胆固醇（一种已知的β -分泌酶处理刺激物）对BACE复合物水平和活性的影响。在目标3中，将评价复合物中BACE活性增强的基础。

DESCRIPTION (provided by applicant): Amyloid deposited in the brains of Alzheimer's disease (AD) patients is composed of a 4kDa amyloid beta peptide or A-beta. A-beta is produced by sequential cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases. A novel type-1 integral membrane aspartyl protease BACE has been identified as the beta-secretase, and knock-out mouse models demonstrate that this enzyme is necessary for A-beta production in the mouse brain. The generation of A-beta by beta-secretase is a minor pathway of APP processing, resulting in 10% of APP processing to its secreted derivatives. Since BACE is a poorly active enzyme in vitro, it has been proposed that its activity is limiting in the cell. However, A-beta production increases by several orders of magnitude in transfected cells expressing high levels of APP without a corresponding increase in BACE activity. To explain these discrepant observations, the PI proposes the hypothesis that APP processing by BACE occurs in a specialized cellular compartment where its activity is not limiting. Instead, only a small pool of APP enters this compartment, accounting for the limiting processing of APP by BACE. This hypothesis is consistent with recent findings from the PI's laboratory indicating that BACE can be isolated as highly active high molecular weight complex from guinea pig brain. To further test this hypothesis, in aim 1 APP chimeras will be generated to target APP to specialized cellular compartments, and its processing will be examined. In aim 2, the effects of cholesterol, a known stimulator of beta-secretase processing, will be evaluated on the levels and activity of BACE complex. In aim 3, the basis of enhanced BACE activity in the complex will be evaluated.