APOE STRUCTURE FUNCTION AND ALZHEIMERS DISEASE

载脂蛋白E是胆固醇转运的重要蛋白质 一种由多种组织合成的系统。在分泌之后， 载脂蛋白被结合在各种脂蛋白颗粒(乳杆菌)中 残留物、极低密度脂蛋白、低密度脂蛋白和高密度脂蛋白的亚组分)，并指导它们的分解代谢 通过细胞受体。在载脂蛋白E中发现了常见的基因突变 这导致了三个纯合子(E4/4，E3/3和E2/2)和三个 杂合型(E4/3、E3/2和E4/2)。载脂蛋白E4含有 发病晚的患者中表型出现的频率增加 阿尔茨海默病(AD)。这是我们的假设，基于现有的 实验数据和模型表明，载脂蛋白E与 脑中分泌的(Abeta)或细胞内蛋白(tau，细胞骨架 蛋白质)依赖于三维排列的改变和 两亲性氨基端apoE螺旋与ApoE的稳定相互作用 它们与羧基末端区域的相互作用被预测为 包含相似的螺旋束。突变可以扰乱 载脂蛋白E的构象可能影响其理化和功能 在体外的性质，并可能导致体内的AD。 为了验证这一假设，我们提出了以下具体目标：1) 对E3基因进行诱变，并通过导入和选择产生 表示变种E形式的线条。六类突变有 经过精心挑选，以破坏静电或疏水 稳定载脂蛋白E螺旋2、3和4以及 ApoE的羧基末端结构域被预测含有类似的 螺旋束。2)大规模种植正常和变异载脂蛋白E形式 (生物反应器滚筒瓶)和纯化突变蛋白 理化和功能分析及毒性用的培养液 神经细胞培养的测试。载脂蛋白E突变型的构象 以及Abeta和apoE的相互作用将由 物理化学方法。选定的构造将通过以下方式引入 在神经细胞培养中进行电穿孔或转染法 评估载脂蛋白E对神经细胞生长、伸展和分支的影响 以及其他形态和细胞骨架的变化。3)学习 不同形式载脂蛋白E的热力学稳定性以及 载脂蛋白E与Abeta和tau的相互作用 对于维护次要设备和 这些相互作用需要三级构象。4)生成 阿尔茨海默病动物模型a)通过表达apoE4基因和 APP751(瑞典突变Met 1产生Leu，Lys-2产生Asn) ApoE基因缺陷小鼠在星形胶质细胞特异性和特异性基因调控下的研究 神经元特异性启动子。为了进行比较，将生成一条鼠标线 ApoE3表达正常，APP751正常。B)通过研究大脑的变化 发生在表达载脂蛋白E和APP的小鼠系中。了解 决定载脂蛋白E构象的结构元素是必不可少的 了解载脂蛋白E的正常和异常功能及其在AD中的作用。

Apolipoprotein E is an important protein of the cholesterol transport system which is synthesized by a variety of tissues. Following secretion, apoE is incorporated in a variety of lipoprotein particles (chylomicron remnants, VLDL, IDL and a subfraction of HDL) and directs their catabolism by cell receptors. Common genetic mutations have been identified in apoE which give rise to three homozygous (E4/4, E3/3 and E2/2) and three heterozygous (E4/3 E3/2 and E4/2) phenotypes. The apo E4 containing phenotypes occur with increased frequency in patients with late onset of Alzheimer's Disease (AD). It is our hypothesis based on existing experimental data and models, that the abnormal interactions of apoE with secreted (Abeta) or intracellular proteins in the brain (tau, cytoskeletal protein) depend on alterations int he three-dimensional arrangement and stabilizing interactions of the amphipathic aminoterminal apoE helices and their interaction with the carboxy terminal domain which is predicted to contain similar helical bundles. Mutations which can disrupt the conformation of apoE may affect its physiochemical and functional properties in vitro and may lead to AD in vivo. To test this hypothesis we propose the following specific aims; 1) to mutagenize the E3 gene and generate by transfection and selection will lines which express the variant E forms. Six categories of mutations have been carefully selected to disrupt the electrostatic or hydrophobic interactions which stabilize helices 2,3 and 4 of apoE as well as the carboxy terminal domain of apoE which is predicted to contain similar helical bundles. 2) To grow the normal and variant apoE forms on a large scale (Bioreactor roller bottles) and purify the mutant proteins from the culture media for physicochemical and functional analyses and toxicity tests on neuronal cell cultures. The conformation of the mutant apoE forms as well as of the Abeta and apoE interactions will be monitored by physicochemical methods. Selected constructs will be introduced by electroporation or transfection in neuronal cell cultures in order to assess the effect of apoE on neuronal cell growth, extension and branching as well as on other morphological and cytoskeletal changes. 3) To study the thermodynamic stability of the variant forms of apoE as well as the interactions of apoE with Abeta and tau in order to establish the molecular details which are important for the maintenance of the secondary and tertiary conformation required for these interactions. 4) To generate animal models of Alzheimer's disease a) by expressing the apoE4 gene and the APP751 (Swedish mutation Met 1 yields Leu, Lys-2 yields Asn) in the apoE deficient mouse strain under the control of astrocyte specific and neuronal specific promoters. For comparison a mouse line will be generated expressing apoE3 and normal APP751. b) by studying the brain changes occurring in mouse lines expressing apoE and APP. Understanding the structural elements of apoE which determine its conformation is essential to understand athe normal and aberrant function of apoE and its role in AD.