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MOLECULAR BASIS OF GAMMA-SECRETASE IN GENERATING AB-PRO

生成 AB-PRO 的伽马分泌酶的分子基础

基本信息

批准号：
2706029
负责人：
RONG WANG
金额：
$ 8.25万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-07-01 至 1999-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2706029
关键词：
Golgi apparatus amyloid proteins endopeptidases endoplasmic reticulum enzyme mechanism enzyme substrate immunoprecipitation mass spectrometry monoclonal antibody point mutation protein biosynthesis protein localization protein sequence site directed mutagenesis tissue /cell culture transfection western blottings

项目摘要

Recent progress in elucidating the pathogenesis of Alzheimer's disease (AD) has centered on the apparent role of the 40-42 residue amyloid beta- peptide (Abeta) as a unifying pathological feature of the genetically diverse forms of this complex disorder. For example, DNA mutations have been identified in several distinct genes (four of which have been discovered to date) associating with familial AD (FAD). In addition, others have shown that the proteolytic processing of beta-amyloid precursor protein (betaAPP) is altered by many of these mutations in a way that results in increased production of Abeta peptides, particularly of the highly hydrophobic (and thus amyloidogenic) 42-residue form of Abeta (Abeta/42). Elevated production of Abeta/42 by FAD-linked genetic mutations indicates that the proteolytic cleavage of betaAPP by gamma- secretase may be a key element in the formation of amyloidogenic form of Abeta. A full understanding of the molecular basis of the substrate specificity of gamma-secretase requires knowledge of how FAD-associated genetic mutations affect the processing of betaAPP in AD. However, none of these enzymes responsible for the proteolytic cleavage of betaAPP have yet been identified. The objective of this study is to investigate the molecular mechanisms of gamma-secretase in the processing of betaAPP, with three specific aims. (1) Characterizing the substrate specificity of gamma-secretase cleavage of betaAPP. (2) Investigating the effects of the association of the transmembrane domain of betaAPP with membrane on gamma- secretase cleavage. (3) Investigating the effects of the localization of betaAPP to different cellular compartment on the function of gamma- secretase. Site directed mutagenesis will be used to vary either the properties of amino acid residues around the gamma-secretase cleavage site or to vary the length of the transmembrane domain of betaAPP. The exact identities of secreted Abeta-related peptides will be determined be measuring their molecular basses using immunoprecipitation and mass spectrometry. These studies will reveal fine detains concerning the cleavage specificities of gamma-secretase towards betaAPP, leading to better strategies to identify the as yet undiscovered enzyme(s).

阿尔茨海默病发病机制的研究进展 (AD)集中在40-42残基淀粉样β蛋白的明显作用- 多肽(Abeta)作为遗传性糖尿病的统一病理特征这种复杂的疾病有多种形式。例如，DNA突变具有在几个不同的基因中被鉴定(其中四个已经被迄今发现)与家族性AD(FAD)有关。此外, 其他研究表明，β-淀粉样蛋白的蛋白质分解过程前体蛋白(Betaapp)在某种程度上会因这些突变而改变。这导致Abeta多肽的产生增加，特别是高度疏水(从而导致淀粉样变性)的42-残基形式的Abeta (Abeta/42)。FAD连锁遗传提高Abeta/42的产量突变表明，γ-APP对β-APP的蛋白水解性切割分泌酶可能是淀粉样变性形式形成的关键因素阿贝塔。充分了解底物的分子基础伽马分泌酶的特异性需要了解FAD如何关联基因突变影响阿尔茨海默病患者β-APP的加工。然而，没有一个这些负责β-APP蛋白水解酶的酶尚未已被确认身份。这项研究的目的是调查 β-分泌酶在β-APP加工过程中的分子机制三个具体目标。(1)底物专一性的表征 β-APP的伽马分泌酶裂解。(2)调查以下情况的影响 β-APP跨膜区与膜结合的研究分泌酶分裂。(3)调查本地化的影响 β-APP对不同细胞亚群伽马-α功能的影响分泌酶。定点突变将被用来改变 γ-分泌酶裂解位点周围氨基酸残基的性质或改变BetaAPP跨膜结构域的长度。完全相同的分泌的Abeta相关多肽的身份将被确定用免疫沉淀法和质量法测定它们的分子碱基光谱分析。这些研究将揭示关于 γ-分泌酶对β-APP的切割特异性，导致确定尚未发现的酶的更好策略(S)。