PROTEIN STRUCTURE IN APOE4-ASSOCIATED NEURODEGENERATION

APOE4 相关神经变性中的蛋白质结构

基本信息

批准号：
7431631
负责人：
KARL WEISGRABER
金额：
$ 35.05万
依托单位：
J. DAVID GLADSTONE INSTITUTES
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-06-01 至 2008-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7431631
关键词：
Abbreviations Address Adverse effects Affect Alkanesulfonates Alzheimer&apos s Disease Amyloid beta-Protein Precursor Apolipoprotein E Apolipoproteins Behavior Binding Biochemical Biological Models Brain Characteristics Chemicals Circular Dichroism Cognitive Condition Cysteine Dimyristoylphosphatidylcholine Disease Disulfides Electron Spin Resonance Spectroscopy Engineering Enzyme-Linked Immunosorbent Assay Ethylenediamine Ethylenediamines Family Figs - dietary Fluorescein Fluorescein-5-isothiocyanate Fluoresceins Fluorescence Resonance Energy Transfer Foundations Functional disorder Funding Gene Targeting Genes Glial Fibrillary Acidic Protein High Density Lipoproteins Homo Homodimerization Human Human Characteristics In Vitro Indiana Individual Isothiocyanates Knock-in Mouse Knowledge Laboratories Link Lipid Binding Lipoproteins Low Density Lipoprotein Receptor Low-Density Lipoproteins Maintenance Membrane Methionine Modeling Molecular Mus Mutation Neomycin resistance gene Nerve Degeneration Neuraxis Neurites Neurons Pathway interactions Pharmaceutical Preparations Phenotype Physical Chemical Technique Play Polyacrylamide Gel Electrophoresis Positioning Attribute Predisposition Process Property Protein Chemistry Protein Isoforms Proteins Relative (related person)Research Personnel Resistance Risk Factors Role Site-Directed Mutagenesis Sodium Spectroscopy, Fourier Transform Infrared Structural Models Structure Study Section Sulfides Testing Transgenic Organisms Translating V717F Very low density lipoprotein amyloid peptide apolipoprotein E-3 apolipoprotein E-4 base design dimer drug development familial Alzheimer disease in vivo in vivo Model light scattering lipid transport member methanethiosulfonate molecular mass mouse model mutant novel novel therapeutics oxidation programs protein function protein misfolding protein structure receptor receptor binding repaired response to injury therapeutic target

项目摘要

Apolipoprotein E (apoE) displays critical isoform-specific effects in neurodegeneration and in the normal maintenance and repair of neurons. Unlike the other major human isoforms, apoE3 and apoE2, apoE4 is an established risk factor for Alzheimer's disease (AD). However, the basis underlying this isoform-specific effect is unknown and, most importantly, has not been explored systematically in terms of the effect of structure on function. A basic paradigm of protein chemistry is that the structure and biophysical properties of a protein determine whether it functions normally or abnormally. Thus, analyzing the structural and biophysical differences among the isoforms can provide important clues regarding the apoE isoform-specific mechanisms and basis for the association of apoE4 with AD. Previous studies fi'om the Project Leader's laboratory identified three major characteristics that distinguish apoE4 from apoE3 and apoE2: (1) the amino-terminal domain of apoE4 is the least resistant to chemical or thermal unfolding and forms a stable folding intermediate, which we determined is a molten globule state; (2) apoE4 lacks cysteine and does not form a disulfide-linked homodimer, whereas apoE3 and apoE2 contain cysteine at position 112 and form dimers; and (3) apoE4 domain interaction, an interaction of the amino- and carboxyl-terminal domains that is unique to apoE4. Our central hypothesis is that one or more of these structural or biophysical differences plays a major role in the association of apoE4 with neurodegeneration or deficits in neuronal repair. Our experimental approach is to alter the mouse Apoe gene by gene targeting to "humanize" mouse apoE with respect to each of the human isoform structural differences by introducing mutations that engineer in these structural differences individually and selectively. Using mouse models expressing mutant apoE displaying selected structural and biophysical features of human apoE4, we will examine the relative contribution of each of the human isoform structural differences to apoE4 behavior. As proof of principle, we have generated a mouse model of apoE4 domain interaction by gene targeting and are characterizing its phenotype. In this proposal, we will extend this structure-based approach with three specific aims that will test the hypothesis that the propensity of apoE4 to form a molten globule state and its lack of cysteine also contribute to the apoE4-specific effects. The identification of the key apoE4 structural and biophysical differences responsible for neurodegeneration holds the potential to provide new opportunities for novel therapeutic strategies designed to interfere with or diminish the pathological impact of these differences.

载脂蛋白E（APOE）在神经变性以及神经元的正常维持和修复中显示出关键的同工型特异性作用。与其他主要的人类同工型APOE3和APOE2不同，APOE4是阿尔茨海默氏病（AD）的既定风险因素。但是，该同工型特异性效应的基础尚不清楚，最重要的是，尚未根据结构对功能的影响进行系统的探索。蛋白质化学的基本范例是，蛋白质的结构和生物物理特性决定了它是正常或异常起作用的。因此，分析同工型之间的结构和生物物理差异可以提供有关APOE同工型特异性机制的重要线索，以及APOE4与AD的关联的基础。该项目负责人的实验室先前的研究确定了将APOE4与APOE3和APOE区分开的三个主要特征。（2）APOE4缺少半胱氨酸，不形成二硫键键合二聚体，而APOE3和APOE2在位置112处含有半胱氨酸并形成二聚体；（3）APOE4域相互作用，这是APOE4独有的氨基和羧基末端域的相互作用。我们的中心假设是，这些结构或生物物理差异中的一个或多个在APOE4与神经变性或神经元修复中的缺陷中起着重要作用。我们的实验方法是通过靶向对每个人类同工型结构差异“人性化”小鼠APOE来改变小鼠APOE基因，通过引入分别和选择性地在这些结构差异中进行工程的突变。使用表达突变体APOE的小鼠模型，显示人类APOE4的选定结构和生物物理特征，我们将研究每个人类同工型结构差异对APOE4行为的相对贡献。作为原理的证明，我们通过基因靶向生成了APOE4结构域相互作用的小鼠模型，并正在表征其表型。在此提案中，我们将通过三个特定目标扩展了这种基于结构的方法，该方法将检验以下假设：APOE4倾向形成熔融球状状态，并且缺乏半胱氨酸也有助于APOE4特异性效应。对神经退行性的关键APOE4结构和生物物理差异的识别具有为新的治疗策略提供新的机会，以干扰或减少这些差异的病理影响。