Regulation and Cell Biology of Beta-Secretase

描述（由申请人提供）：阿尔茨海默病（AD）患者脑中沉积的淀粉样蛋白由4kDa淀粉样蛋白β肽或A-β组成。A-β是由β-和γ-分泌酶连续切割淀粉样前体蛋白（APP）产生的。一种新的1型整合膜乙酰基蛋白酶BACE已被鉴定为β-分泌酶，敲除小鼠模型证明这种酶是小鼠脑中A-β产生所必需的。通过β-分泌酶产生A-β是APP加工的次要途径，导致10%的APP加工成其分泌衍生物。由于BACE在体外是活性差的酶，因此已经提出其活性在细胞中是有限的。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活性没有相应的增加。为了解释这些不一致的观察结果，PI提出了一个假设，即BACE对APP的加工发生在其活性不受限制的专门细胞区室中。相反，只有一小部分APP进入这个隔间，这说明BACE对APP的处理有限。该假设与PI实验室的最新发现一致，表明BACE可作为高活性高分子量复合物从豚鼠脑中分离。为了进一步检验这一假设，在目的1中，将产生APP嵌合体以将APP靶向至专门的细胞区室，并且将检查其加工。在目标2中，将评价胆固醇（一种已知的β-分泌酶加工刺激剂）对BACE复合物水平和活性的影响。在目标3中，将评估复合物中增强的BACE活性的基础。

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.