Structural characterization of APP family proteins

项目总结/摘要 阿尔茨海默病（Alzheimer disease，AD）是一种以脑功能进行性衰退为特征的神经退行性疾病 功能、突触进行性丧失、神经元死亡、脑萎缩和细胞外淀粉样蛋白形成 斑块和细胞内神经纤维缠结。淀粉样前体蛋白（APP）是一种I型淀粉样前体蛋白， 在AD发病机制中起核心作用的跨膜糖蛋白，作为β-淀粉样蛋白的前体， 淀粉样肽（Aβ）是淀粉样斑块的主要成分。此外，APP上的几个突变 基因与早发性遗传性阿尔茨海默病（家族性阿尔茨海默病或FAD）有关。 APP除了是毒性Aβ肽的前体和FAD突变的载体外， 与广泛的生物学功能相关，包括增殖，神经元发育， 细胞内运输，Ca 2+稳态，神经突生长，树突复杂性的形成，以及大多数 重要的是，突触发生和突触功能。的确，多份报告将APP与脊柱调节联系起来 密度、棘动力学和突触可塑性。已报道的APP在突触前活动中的突触作用 区和突触后密度包括调节突触前烟碱受体，促进 神经递质的释放、突触囊泡再循环的调节和细胞表面表达的调节 和NMDA受体的活性。因此，放松对上述一项或多项职能的管制可能会导致 突触功能障碍和神经变性，也是AD的基本标志。APP是基因家族的一部分， 与另外两个哺乳动物成员，淀粉样前体蛋白1（APLP 1）和淀粉样蛋白 前体样蛋白2（APLP 2）。两种APLPs与APP高度同源，具有相似的分子结构， 结构和功能，特别是与突触发生，脊柱动力学和突触可塑性。的 本提案的目标是开发合适的实验资源，用于进行结构研究， APP和APLPs的生理功能。为了实现这一目标，我们建议建立一个表达式 和纯化管道，用于APP、APLP 1和APLP 2直向同源物的中等通量筛选，研究 一个替代的标记程序和比较提取和重建方法的直系同源物的子集。 最后，我们建议利用单粒子低温电子显微镜（cryoEM）来阐明原子水平的 APP家族蛋白分子结构的细节。一个完整的高分辨率结构信息- APP家族蛋白的长度将使我们更好地了解生理功能的结构基础 APP和APP样蛋白质，并告知我们的理解，这些功能是如何在AD失调。

Project Summary/Abstract Alzheimer disease (AD) is a neurodegenerative disease characterized by progressive decline of brain functions, progressive loss of synapses, neuronal death, brain atrophy and formation of extracellular amyloid plaques and intracellular neurofibrillary tangles. The amyloid precursor protein (APP) is a type I transmembrane glycoprotein which plays a central role in AD pathogenesis, as the precursor of the beta amyloid peptide (Aβ), the main component of amyloid plaques. In addition, several mutations on the APP gene are associated with the early-onset hereditary form of the disease (Familial Alzheimer disease or FAD). Apart from being the precursor of the toxic Aβ peptide and the carrier of FAD mutations, APP has been associated with a wide range of biological functions, including proliferation, neuronal development, intracellular transport, Ca2+ homeostasis, neurite outgrowth, shaping of dendritic complexity and, most importantly, synaptogenesis and synaptic function. Indeed, multiple reports link APP to the regulation of spine density, spine dynamics and synaptic plasticity. Reported synaptic roles of APP both in the presynaptic active zone and postsynaptic density include regulation of presynaptic nicotinic receptors, facilitation of neurotransmitter release, regulation of synaptic vesicle recycling and regulation of the cell surface expression and activity of NMDA receptors. Deregulation of one or more of the above functions is therefore likely to lead to synaptic dysfunction and neurodegeneration, also basic hallmarks of the AD. APP is part of a gene family, with two more mammalian members, the Amyloid Precursor Like Protein 1 (APLP1) and the Amyloid Precursor Like Protein 2 (APLP2). Both APLPs are highly homologous to APP, with similar molecular architecture and functions, especially related to synaptogenesis, spine dynamics and synaptic plasticity. The goal of this proposal is to develop suitable experimental resources for performing structural investigations of the physiological functions of APP and APLPs. To achieve this goal, we propose to establish an expression and purification pipeline for medium throughput screening of APP, APLP1 and APLP2 orthologs, investigate an alternative tagging procedure and compare extraction and reconstitution methods for a subset of orthologs. Finally, we propose to utilize single particle cryo-electron microscopy (cryoEM) to elucidate atomic-level details of the molecular architecture of APP family proteins. High-resolution structural information of a full- length APP family protein will enable us to better understand the structural basis of the physiological functions of APP and APP-like proteins and inform our understanding of how these functions are deregulated in AD.