Protein Structure in ApoE4-associated Neurodegeneration

描述(申请人提供)：载脂蛋白E(ApoE)在神经退行性变和神经元的正常维护和修复中显示出关键的异构体特异性作用。与人类其他主要亚型apoE3和apoE2不同，apoE4是阿尔茨海默病(AD)的既定风险因素。然而，这种异构体特异性效应的基础尚不清楚，最重要的是，还没有系统地从结构对功能的影响方面进行探索。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它的功能是正常的还是异常的。因此，分析不同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的关联提供基础。PI实验室先前的研究确认了apoE4有别于apoE3和apOE2的三个主要特征：(1)apoE4的氨基末端结构域最不耐化学或热展开，并形成稳定的折叠中间体，我们确定它是一个熔融的球状结构；(2)apoE4缺乏半胱氨酸，不形成二硫键连接的同源二聚体，而apoE3和APOE2在112位含有半胱氨酸并形成二聚体；以及(3)apoE4结构域的相互作用，即apoE4所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与神经退行性变或神经元修复缺陷的关联中发挥了重要作用。我们的实验方法是通过单独和选择性地引入突变来设计这些结构差异，从而逐个基因地改变小鼠APOE的靶向，使小鼠APOE相对于每个人类异构体结构差异“人性化”。使用表达突变apoE的小鼠模型显示选定的人类apoE4的结构和生物物理特征；我们将检查每个人类异构体结构差异对apoE4行为的相对贡献。作为原理的证明，我们已经通过基因打靶建立了apoE4结构域相互作用的小鼠模型，并正在表征其表型。在这项提案中，我们将扩展这种基于结构的方法，有三个具体的目标，这将检验这一假设，即apoE4形成熔融球状状态的倾向和它的半胱氨酸的缺乏也有助于apoE4的特异性效应。对导致神经退行性变的关键apoE4结构和生物物理差异的识别有可能为干扰或减少这些差异的病理影响的新的治疗策略提供新的机会。

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.