STRUCTURAL AND FUNCTIONAL ANALYSIS OF PRESENILINS

描述(摘自申请者摘要)：阿尔茨海默病(AD)，最常见的 老年人常见的进行性痴呆症，其特点是 β淀粉样多肽(Abeta)在脑实质和脑内的沉积 脑血管。AD的一个子集，分类为家族性早发性AD(FAD)， 是一种常染色体显性遗传病。编码基因的突变 多表位膜蛋白，称为早老素1(PS1)和早老素2(PS2)， 占早发性阿尔茨海默病的大多数。早衰者(PS)扮演一个 在Abeta多肽的产生中起重要作用。阿贝塔产品是 在PS1缺失(PS-1-)细胞中被取消。此外，FAD连锁突变体PS1 增加高纤维蛋白原Abeta42多肽的产量。精准的 PS1在Abeta产生中的作用及其分子机制 FAD相关的PS1突变导致Abeta42产量上升尚未得到证实 已定义。了解这些问题对AD研究至关重要。 我们认为，PS1的分子和结构域分析将 提供对清楚了解基因突变如何 可能影响PS1的正常功能(S)，并赋予致病 突变型PS1多肽的性质。目前，人们对此知之甚少 关于PS1的分子和结构域。为了解决这个问题， 我们将产生一系列含有实验性缺失的PS1多肽 并评估对：PS1内切蛋白降解、“γ-分泌酶”处理的影响 淀粉样前体蛋白，以及Notch1的膜内裂解 作为评估含有FAD连接的缺失多肽的潜力 错义突变导致Abeta42水平升高。 已知PS1的表达在翻译后受到严格调控 通过与其他蛋白质形成复合体而达到水平；然而其机制(S) 对这一规定负责的人还没有定义。为了获得洞察力 关于PS1蛋白积累的调节，我们将进行一项 基于新型逆转录病毒表达克隆策略的功能筛选 确定参与调节PS1积累的蛋白质。因为 在没有PS1的情况下，很少或不产生Abeta，即蛋白质的同一性 调节PS水平对于设计合理的治疗方案至关重要 旨在减少Abeta负担的战略。 最后，我们概述了转基因策略，以检查在体内的作用 PS1的亲水结构域，是PS1之间保守程度最低的结构域 和PS同系物。最近的研究预测了这一点的重要作用(S 基于磷酸化、caspase裂解和蛋白质相互作用的结构域。 我们的努力将集中在PS1亲水环结构域在 哺乳动物胚胎发育，并在淀粉样蛋白的过程中 转基因小鼠大脑中的生产/沉积。

DESCRIPTION (From the Applicant's Abstract): Alzheimer's disease (AD), the most common type of progressive dementia in the elderly, is characterized by the deposition of beta-amyloid peptides (Abeta) in the brain parenchyma and cerebral vessels. A subset of AD, classified as familial early-onset AD (FAD), is inherited as an autosomal dominant disorder. Mutations in genes encoding polytopic membrane proteins, termed presenilin 1 (PS1) and presenilin 2 (PS2), account for the majority of early-onset cases of AD. Presenilins (PS) play an important role in the generation of Abeta peptides. Abeta production is abrogated in PS1-deficient (PS-1-) cells. Moreover, FAD-linked mutant PS1 increases the production of highly fibrillogenic Abeta42 peptides. The precise role of PS1 in Abeta production, and the molecular mechanisms by which FAD-linked PS1 mutations lead to elevations in Abeta42 production have not been defined. Understanding these issues is of central importance to AD research. It is our view that molecular and structural domain analysis of PS1 will provide information critical for a clear understanding of how genetic mutations in PS1 might influence the normal function(s) of PS1, and confer pathogenic properties to mutant PS1 polypeptides. At present, very little is known regarding the molecular and structural domains of PS1. To address this issue, we will generate a series of PS1 polypeptides harboring experimental deletions and assess the influence on: PS1 endoproteolysis, "gamma-secretase" processing of amyloid precursor protein, the intramembranous cleavage of Notch1, as well as evaluate the potential of the deletion polypeptides harboring FAD-linked missense mutations to elevate the levels of Abeta42. It is known that PS1 expression is tightly regulated at the post-translational level by complex formation with other proteins; however the mechanism(s) responsible for this regulation have not been defined. To gain insights regarding the regulation of PS1 protein accumulation, we will perform a functional screen based on a novel retroviral expression cloning strategy to identify proteins that participate in regulating PS1 accumulation. Because little or no Abeta is produced in the absence of PS1, identity(ies) of proteins that regulate PS levels is critical for the design of rational therapeutic strategies aimed at reducing Abeta burden. Finally, we have outlined transgenic strategies to examine the in vivo role of the hydrophilic domain of PS1, which is the domain least conserved between PS1 and PS homologues. Recent studies have predicted important function(s) for this domain based on phosphorylation, caspase cleavage, and protein interactions. Our efforts will focus on the role played by PS1 hydrophilic loop domain during mammalian embryonic development, and in the process of amyloid production/deposition in the brains of transgenic mice.