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，是由分泌酶和分泌酶对淀粉样蛋白前体（APP）的顺序切割形成的。？分泌酶在？处切割APP跨膜结构域地点，产生A¿的c端，在？产生APP胞内结构域的n端。这个项目的目标是理解？——分泌酶复合体将长A肽中间体修剪成更短的形式，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)哪些较短的A¿肽是通过修剪特定的长A¿肽产生的？我们将鉴定和量化所有由正常？乳沟产品A¿49和A¿48。我们还将确定A¿50，这不是一个？正常解理产物，导致形成其他未观察到的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，这个规则有多严格。(2)如何？-分泌酶完成长A肽中间体的c端三肽修剪。证据支持最初的？蛋白质水解产生长A -肽，然后每3个残基切割，但其发生的机制尚不清楚。我们假设新形成的长A¿的羧基末端产生于？乳沟靠什么？-分泌酶对每3个残基的修剪和明显的精度至关重要。为了验证这一假设，我们将研究长A¿肽的c端酰胺作为底物的能力。(3)引起阿尔茨海默病的PS1突变对长A¿肽修剪的影响是什么？我们已经证明这种ps1突变体？-分泌酶复合物可以增加重组APP底物中长-短A¿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突变通常通过减缓修剪过程来增加长A¿肽的比例。

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.