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显示出关键的同种型特异性作用。与其他主要的人类同种型apoE 3和apoE 2不同，apoE 4是阿尔茨海默病（AD）的既定危险因素。然而，这种异构体特异性效应的基础是未知的，最重要的是，还没有系统地探讨结构对功能的影响。蛋白质化学的一个基本范式是蛋白质的结构和生物物理性质决定了它是否正常或异常地发挥功能。因此，分析异构体之间的结构和生物物理差异，可以提供重要的线索，关于apoE异构体的具体机制和基础的apoE 4与AD的关联。PI实验室先前的研究确定了apoE 4与apoE 3和apoE 2的三个主要特征：（1）apoE 4的氨基端结构域对化学或热解折叠的抵抗力最小，并形成稳定的折叠中间体，我们确定其为熔融球;（2）apoE 4缺乏半胱氨酸，不形成二硫键连接的同源二聚体，而apoE 3和apoE 2在112位含有半胱氨酸，形成二聚体;和（3）apoE 4结构域相互作用，apoE 4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apoE 4与神经元变性或神经元修复缺陷的关联中起主要作用。我们的实验方法是改变小鼠ApoE基因的基因靶向，以“人源化”小鼠apoE相对于每个人的亚型结构差异，通过引入突变，工程师在这些结构差异个别和选择性。使用小鼠模型表达的突变体apoE显示选定的结构和生物物理特征的人apoE 4，我们将检查每个人的亚型结构差异apoE 4的行为的相对贡献。作为原理的证明，我们已经通过基因靶向产生了apoE 4结构域相互作用的小鼠模型，并表征其表型。在这个提议中，我们将扩展这种基于结构的方法，有三个具体的目标，将测试的假设，即apoE 4的倾向，形成一个熔融的球状态和缺乏半胱氨酸也有助于apoE 4特异性的影响。负责神经退行性变的关键apoE 4结构和生物物理差异的鉴定具有为设计用于干扰或减少这些差异的病理影响的新型治疗策略提供新机会的潜力。

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.