MOLECULAR AND FUNCTIONAL ANALYSIS OF HUMAN APOA-I

具体目标。载脂蛋白A-I（apo-A-I）是主要的蛋白质成分 高密度脂蛋白（HDL），并作为酶的激活剂 卵磷脂胆固醇酰基转移酶（LCAT）。 的长期目标 这一建议是为了了解结构与功能的关系， 人apoA-I。 在这个应用程序中，重点将放在域上 和蛋白质上的残基，其1）参与活化 LCAT，和2）在稳定A-I构象方面都是重要的， 在溶液中并与脂质结合。 这是我们的假设，基于 现有的实验数据和模型，apoA-I的能力， 以盘状磷脂双层的形式与脂质结合 复合物或球形HDL和激活LCAT取决于三个- 两亲性螺旋重复序列的三维排列。 具体 目标是：1.为了诱变人类apo-A-I基因，通过 转染和选择表达不同形式的 apoA-I，并从培养基中分离蛋白质用于结构分析。 和功能分析使用已建立的方法。 选择 将基于三个标准：i） 某些apoA-I结构域用于LCAT活化和脂质结合， 实验数据和现有模型。（二）收费优惠 可能起作用的拓扑学特异性位置的氨基酸 在稳定并排相互作用静电相互作用中 apoA-I中的α-螺旋在溶液中和与脂质的结合中; iii）亮氨酸拉链基序和特定氨基酸序列中的其他残基的位置， 在α-螺旋中的位置，这可能稳定的三级折叠， A-I在溶液中呈螺旋束状或可能参与缔合 与脂质。 2. 研究这些变体的热力学稳定性 apoA-I的形式，以建立分子细节 对维持二级和三级构象很重要 apoA-I在溶液和细胞中的功能作用所需的 与脂类的联系。 3. 为了研究的功能特性， apoA-I突变体特别形成了它们激活LCAT的能力， 与磷脂胆固醇囊泡、模型脂肪乳剂结合， HDL。 LCAT活化和脂质结合的变化将被 与apoA-I的预测或观察到的改变相关 结构 具有低或高血浆apoA-I或HDL的人类受试者具有低或高血浆apoA-I或HDL， 分别增加或降低发生动脉粥样硬化的风险。 了解apoA-I的结构元件， 二级和三级构象对于理解 这种非常重要的蛋白质的功能和抗动脉粥样硬化特性。

Specific Aims. Apolipoprotein A-I (apo-A-I) is the main protein component of high density lipoprotein (HDL) and acts as an activator of the enzyme lecithin cholesterol acyltransferase (LCAT). The long term objective of this proposal is to understand the structure-function relationships of human apoA-I. In this application emphasis will be placed on the domains and residues on the protein which 1) participate in the activation of LCAT, and 2) are important in stabilizing the conformation of A-I both in solution and in association with lipid. It is our hypothesis, based on existing experimental data and models, that the ability of apoA-I to associate with lipid in the form of discoidal phospholipid bilayer complexes or spherical HDL and activate LCAT depends on the three- dimensional arrangement of the amphipathic helical repeats. The specific aims are: 1. To mutagenize the human apo-A-I gene, generate by transfection and selection cell lines which express variant forms of apoA-I, and isolate the proteins from the culture media for structural and functional analysis using established methodologies. The selection of the mutations will be based on three criteria: i) The importance of certain apoA-I domains for LCAT activation and lipid binding as suggested by experimental data and existing models. ii) The preference of charged amino acids for topologically specific positions which may play a role in electrostatic interaction that stabilize the side-by-side interactions of a-helices in apoA-I both in solution and in association with lipids; iii) the location of leucine zipper motifs and other residues in specific positions in the a-helices, which may stabilize the tertiary folding of A-I in solution in a helical bundle or may be involved in the association with lipids. 2. To study the thermodynamic stability of these variants forms of apoA-I with a view to establishing the molecular details important for the maintenance of the secondary and tertiary conformation required for the functional roles of apoA-I both in solution and in association with lipids. 3. To study the functional properties of the mutants apoA-I forms particularly their ability to activate LCAT and to bind to phospholipid cholesterol vesicles, model lipid emulsions and to HDL. Changes in LCAT activation and lipid binding will then be correlated with predicted or observed alterations in the apoA-I structure. Human subjects with low or high plasma apoA-I or HDL have an increased or decreased risk respectively of developing atherosclerosis. Understanding the structural elements of apoA-I which determine its secondary and tertiary conformation is essential to understand the function and anti-atherogenic properties of this very important protein.