STRUCTURAL AND FUNCTIONAL ANALYSIS OF PRESENILINS

描述（来自申请人的摘要）：阿尔茨海默病（AD），最常见的疾病， 一种常见的老年进行性痴呆，其特征是 β-淀粉样肽（Abeta）在脑实质中的沉积， 脑血管AD的一个子集，被分类为家族性早发性AD（FAD）， 是一种常染色体显性遗传病基因突变编码 多位膜蛋白，称为早老素1（PS1）和早老素2（PS2）， 占AD早发病例的大多数。早老素（PS）发挥作用， 在Abeta肽的产生中起重要作用。Abeta生产 在PS-1-缺陷（PS-1-）细胞中消除。此外，FAD连锁突变体PS1 增加了高原纤性A β 42肽的产生。的精确 PS1在Abeta产生中的作用，以及 FAD连锁的PS1突变导致Abeta 42产生升高， 定义了了解这些问题对AD研究至关重要。 我们认为，PS1的分子和结构域分析将 为清楚地了解基因突变是如何 可能影响PS1的正常功能， 突变型PS1多肽的特性。目前，人们所知甚少 关于PS1的分子和结构域。为了解决这个问题， 我们将产生一系列带有实验性缺失的PS1多肽， 并评估对PS1内蛋白水解、“γ-分泌酶”加工的影响 淀粉样蛋白前体蛋白，Notch 1的膜内切割，以及 作为评估携带FAD-连接的缺失多肽的潜力， 错义突变来提高Abeta 42的水平 已知PS1的表达在翻译后水平受到严格调控， 通过与其他蛋白质形成复合物来提高水平;然而， 这一规定的责任尚未确定。感悟 关于PS1蛋白积累的调节，我们将进行 基于新的逆转录病毒表达克隆策略的功能筛选， 鉴定参与调节PS1积累的蛋白质。因为 在不存在PS1的情况下，很少或不产生Abeta， 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，其是PS1和PS2之间最不保守的结构域， 和PS同系物。最近的研究预测了这一重要功能 结构域基于磷酸化，半胱天冬酶切割和蛋白质相互作用。 我们的努力将集中在PS1亲水环结构域在细胞凋亡过程中所起的作用。 在哺乳动物胚胎发育过程中， 在转基因小鼠的大脑中产生/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.