Trimming of Amyloid Peptides by Gamma-Secretase

描述（由申请人提供）：淀粉样蛋白（A <$）与阿尔茨海默病（AD）的发病机制密切相关，它是由淀粉样蛋白前体（APP）依次被<$-和？-分泌酶?-分泌酶切割APP跨膜结构域在？网站，生产的C-末端的A？，并在？位点，产生APP胞内结构域的N-末端。这个项目的目标是了解这个过程，分泌酶复合体将长的A肽中间体修剪成较短的形式，以及引起疾病的PS突变如何改变活性。考虑到这些目标，将解决以下具体问题：（1）通过修剪特定的长A肽产生哪些较短的A肽？我们将确定和量化所有的A肽形成正常？裂解产物A <$49和A <$48。我们还将确定，如果A UNK 50，这不是一个？正常的裂解产物，导致形成否则观察不到的A <$47，A <$44和A <$41。结果将有助于回答每3个残基修剪是否是一般规则的问题，如果是这样，这个规则有多严格。(2)怎么样？-分泌酶完成长A肽中间体的C末端三肽修剪？有证据支持初次？蛋白水解产生长A肽，然后每3个残基裂解，但发生这种情况的机制尚不清楚。我们假设，新形成的羧基末端的长A？乳沟？分泌酶对于修剪是关键的，并且每3个残基具有明显的精确度。为了验证这一假设，我们将检查长A肽的C-末端酰胺作为底物的能力。(3)导致阿尔茨海默病的PS1突变对长A肽的修剪有什么影响？我们已经表明，这样的PS1突变？-分泌酶复合物可以增加重组APP底物中长肽到短肽的比例。在这里，我们将测试这些突变蛋白酶复合物对A <$49和A <$48的转化，检查形成的A <$产物，这些产物的比例，以及它们相对于野生型复合物的形成速率。我们假设致病的PS1突变通过减缓修剪过程来增加长A肽的比例。 公共卫生相关性：本项目的目标是了解？分泌酶，一种对阿尔茨海默病的病因至关重要的复合酶。生化实验被提出来测试关于这种酶如何工作以及它如何在阿尔茨海默病中失调的假设。

DESCRIPTION (provided by applicant): The amyloid -protein (A¿), strongly implicated in the pathogenesis of Alzheimer's disease (AD), is formed by the sequential cleavage of the amyloid -protein precursor (APP) by ¿- and ?-secretases. ?-Secretases cleaves the APP transmembrane domain at the ? site, producing the C-terminus of A¿, and at the ? site, producing the N-terminus of the APP intracellular domain. The goal of this project is to understand the process by which the ?--secretase complex trims long A¿ peptide intermediates into shorter forms and how disease- causing PS mutations alter activity. With these goals in mind, the following specific questions will be addressed: (1) Which shorter A¿ peptides are produced via the trimming of specific long A¿ peptides? We will identify and quantify all A¿ peptides formed from normal ? cleavage products A¿49 and A¿48. We will also determine if A¿50, which is not a ? normal cleavage product, leads to the formation of the otherwise unobserved A¿47, A¿44 and A¿41. The result will help answer the question of whether trimming by every 3 residues is a general rule, and if so, how rigid is this rule. (2) How does ?-secretase accomplish C-terminal tripeptide trimming of long A¿ peptide intermediates? Evidence supports initial ? proteolysis to produce long A¿ peptides and then cleavage every 3 residues, but the mechanism by which this occurs is unknown. We hypothesize that the newly formed carboxy-terminus of long A¿ produced upon ? cleavage by ?-secretase is critical for trimming and with apparent precision by every 3 residues. To test this hypothesis, we will examine the ability of C-terminal amides of long A¿ peptides to serve as substrates. (3) What are the effects of Alzheimer-causing PS1 mutations on the trimming of long A¿ peptides? We have shown that such PS1-mutant ?-secretases complexes can increase the proportion of long-to-short A¿ peptides from recombinant APP substrate. Here, we will test the conversion of A¿49 and A¿48 by these mutant protease complexes, examining the A¿ products that are formed, the proportion of these products, and their rates of formation relative to the wild-type complex. We hypothesize that the disease-causing PS1 mutations increase the proportion of long A¿ peptides by slowing down the trimming process in general. PUBLIC HEALTH RELEVANCE: The goal of this project is to understand ?-secretase, a complex enzyme critical to the cause of Alzheimer's disease. Biochemical experiments are proposed to test hypotheses about how this enzyme works and how it is dysregulated in Alzheimer's disease.