APOE STRUCTURE FUNCTION AND ALZHEIMERS DISEASE

载脂蛋白E是一种重要的胆固醇转运蛋白 由多种组织合成的系统。 分泌后， 载脂蛋白E掺入各种脂蛋白颗粒（乳糜微粒 残余物、VLDL、IDL和HDL的亚组分），并指导它们的催化剂 细胞受体。 常见的基因突变已被确定在apoE 这产生了三个纯合子（E4/4，E3/3和E2/2）和三个纯合子（E4/4，E3/3和E2/2）。 杂合子（E4/3、E3/2和E4/2）表型。 载脂蛋白E4 表型在迟发性脑梗死患者中发生频率增加， 阿尔茨海默病（AD）。 我们的假设是基于现有的 实验数据和模型表明，apoE与 脑中的分泌蛋白（Abeta）或细胞内蛋白（tau，细胞骨架蛋白 蛋白质）依赖于三维排列的改变， 稳定两亲性氨基末端apoE螺旋的相互作用， 它们与羧基末端结构域的相互作用被预测为 都有类似的螺旋束 突变可以破坏 apoE的构象可能影响其生理化学和功能 在体外的性质，并可能导致AD在体内。 为了验证这一假设，我们提出了以下具体目标：1） 诱变E3基因并通过转染和选择产生， 表示变体E形式的线。 六种类型的突变 经过精心挑选，以破坏静电或疏水 稳定apoE的螺旋2、3和4的相互作用以及 apoE的羧基末端结构域，预测其含有类似的 螺旋束 2)为了在一个大的 规模（生物反应器滚瓶），并纯化突变蛋白从 用于理化和功能分析及毒性的培养基 神经元细胞培养试验。 突变型apoE的构象 以及Abeta和apoE的相互作用将由 物理化学方法 选定的结构将由 电穿孔或转染神经元细胞培养物， 评估apoE对神经元细胞生长、延伸和分支的影响 以及其它形态学和细胞骨架变化。 3)研究 apoE变体的热力学稳定性以及 apoE与Abeta和tau的相互作用，以建立分子 对维护辅助设备和 这些相互作用所需的三级构象。 4)完成网站 a）通过表达apoE 4基因的阿尔茨海默病动物模型， APP 751（瑞典突变Met 1产生Leu，Lys-2产生Asn）在 apoE缺陷小鼠品系在星形胶质细胞特异性和 神经元特异性启动子 为了进行比较，将生成一条鼠标线 表达apoE 3和正常APP 751。B）通过研究大脑的变化 在表达apoE和APP的小鼠品系中发生。 决定apoE构象的结构元件是必不可少的 了解apoE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.