Trimming of Amyloid Peptides by Gamma-Secretase

描述(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，由淀粉样蛋白前体(APP)被β-和β-分泌酶连续切割而形成。？-分泌酶切割APP跨膜区？站点，产生A？的C-末端，并在？位点，产生APP胞内域的N-末端。这个项目的目标是了解分泌酶复合体将长A肽修剪成较短形式的过程，以及致病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？肽来产生哪些较短的A？肽？我们将鉴定和量化所有由正常？切割产物A？49和A？48。我们还将确定A？50，这不是A？正常的切割产物，导致未观察到的A？47、A？44和A？41的形成。这一结果将有助于回答每3个残基修剪一次是否是一般规则，如果是，这一规则有多严格的问题。(2)-分泌酶是如何完成长A肽中间体的C-末端三肽修剪的？有证据支持缩写吗？蛋白质分解产生长的Aβ多肽，然后每3个残基切割一次，但发生这种情况的机制尚不清楚。我们假设新形成的长A‘的羧基末端产生于？分泌酶的切割是修剪的关键，而且每3个残基就能明显精确地切割一次。为了验证这一假设，我们将检验长Aβ多肽的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？结果表明，这种PS1-突变型？-分泌酶复合体可以增加重组APP底物中长-短肽的比例。在这里，我们将测试这些突变的蛋白酶复合体对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体的形成速度。我们假设，致病的PS1突变通过减缓一般的修剪过程来增加长A？肽的比例。 与公共健康相关：该项目的目标是了解分泌酶，这是一种对阿尔茨海默病病因至关重要的复杂酶。生化实验被提议用来测试关于这种酶是如何工作的假说，以及它在阿尔茨海默病中是如何调节失调的。

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.