Protein Structure in ApoE4-associated Neurodegeneration

描述（由申请人提供）：载脂蛋白E （apoE）在神经变性和神经元的正常维持和修复中表现出关键的亚型特异性作用。与其他主要的人类亚型apoE3和apoE2不同，apoE4是阿尔茨海默病（AD）的一个确定的危险因素。然而，这种异构体特异性作用的基础是未知的，最重要的是，还没有系统地探索结构对功能的影响。蛋白质化学的一个基本范例是，蛋白质的结构和生物物理性质决定了它的功能是否正常。因此，分析apoE亚型之间的结构和生物物理差异可以为apoE亚型特异性机制提供重要线索，并为apoE4与AD的关联提供依据。PI实验室先前的研究确定了区分apoE4与apoE3和apoE2的三个主要特征：(1)apoE4的氨基末端结构域对化学或热展开的抗性最低，并形成稳定的折叠中间体，我们确定其为熔融球状体；(2) apoE4缺乏半胱氨酸，不形成二硫连接的二聚体，而apoE3和apoE2在112位含有半胱氨酸，形成二聚体；(3) apoE4结构域相互作用，这是apoE4特有的氨基和羧基末端结构域的相互作用。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缺陷的关联中起主要作用。我们的实验方法是通过单独和选择性地引入突变来改变小鼠Apoe基因的每个基因靶向，使小鼠Apoe相对于每个人类同工异构体结构差异“人源化”。使用表达突变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异构体结构差异对apoE4行为的相对贡献。作为原理证明，我们通过基因靶向建立了apoE4结构域相互作用的小鼠模型，并对其表型进行了表征。在本提案中，我们将扩展这种基于结构的方法，有三个具体目标，将测试apoE4形成熔融球状状态的倾向及其缺乏半胱氨酸也有助于apoE4特异性效应的假设。鉴定出导致神经退行性变的关键apoE4结构和生物物理差异，有可能为设计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。

DESCRIPTION (provided by applicant): 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 effects 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 from the PI'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; (2) apoE4 lacks cysteine and does not form a disulfide-linkedhomodimer, 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 play 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.