Regulation and Cell Biology of Beta-Secretase

描述(申请人提供)：阿尔茨海默病(AD)患者大脑中沉积的淀粉样蛋白由4 kDa的淀粉样β蛋白或A-β组成。A-β是由淀粉样前体蛋白(APP)被β-和伽马分泌酶连续裂解而产生的。一种新的1型整合膜天冬氨酸蛋白酶BACE已被鉴定为β-分泌酶，基因敲除的小鼠模型表明，该酶是小鼠大脑产生A-β所必需的。由β-分泌酶产生的A-β是APP加工的一个次要途径，导致APP加工的10%是其分泌的衍生物。由于BACE在体外是一种活性很低的酶，因此有人认为它在细胞内的活性是有限的。然而，在表达高水平APP的转基因细胞中，A-β的产量增加了几个数量级，而BACE活性却没有相应的增加。为了解释这些不一致的观察结果，PI提出了一种假设，即BACE对APP的处理发生在一个专门的细胞隔间，在那里它的活动不受限制。相反，只有一小部分APP进入这个隔间，这是BACE限制APP处理的原因。这一假设与PI实验室最近的发现一致，即BACE可以作为高活性的高分子复合体从豚鼠脑中分离出来。为了进一步验证这一假设，在AIM 1中，将生成APP嵌合体，以将APP定向到专门的细胞隔间，并检查其处理过程。在目标2中，将对BACE复合体的水平和活性进行评估，胆固醇是一种已知的β-分泌酶加工刺激物。在目标3中，将评估复合体中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.