HIGH DENSITY LIPOROTEIN STRUCTURE-FUNCTION CORRELATIONS

本研究的长期目标是阐明高密度脂蛋白(高密度脂蛋白)的结构与功能之间的关系。在循环中，高密度脂蛋白溶解脂质，从外周细胞中去除胆固醇，激活胆固醇的酯化反应，并将胆固醇酯输送到肝脏和类固醇生成组织进行新陈代谢和排泄。高密度脂蛋白的这些功能，由其主要蛋白成分载脂蛋白A-I(apoA-I)介导，是这类脂蛋白抗动脉粥样硬化作用的基础。在高密度脂蛋白代谢的几个关键步骤中，apoA-I经历了构象变化，以适应高密度脂蛋白中脂类含量的变化，允许apoA-II与高密度脂蛋白结合，并可能介导与膜和卵磷脂胆固醇酰基转移酶(LCAT)的相互作用。在apoA-I的构象变化中存在一个可能的“铰链”区域。该项目的具体目标是确定apoA-I的负责“铰链”功能的螺旋和调节“铰链”结构域构象适应性的关键氨基酸，并研究apoA-I的“封闭”和“开放”铰链形式之间的结构和动力学差异。为了确定apoA-I“铰链”功能所涉及的螺旋，我们建议构建apoA-I的突变体并在大肠杆菌中表达，用apoA-I的第一个或最后一个螺旋替换每个候选螺旋，apoA-I与脂质紧密结合，不能移动。在用光谱学(CD和荧光)方法对突变体进行结构研究并研究它们的脂质结合特性后，将检查定义的RHDL颗粒中的apoA-I突变体的“铰链”功能：颗粒重排、apoA-II结合、与双层膜的相互作用和LCAT激活。通过构建apoA-I的半胱氨酸突变体作为特定的交联点或荧光标记位点，将确认“铰链”螺旋的身份，并研究其结构重排及其动力学。Pro残基在“铰链”螺旋(ES)中的疏水性、电荷分布和作用将通过单个氨基酸的突变替代来评估。这些研究有望定位载脂蛋白A-I的“铰链”区域，并阐明其在载脂蛋白A-I的几个重要功能中的作用机制。

The long-term goal of this research is to elucidate the structure-function relationships in high density lipoproteins (HDL). In circulation, HDL solubilizes lipids, removes cholesterol from peripheral cells, activates the esterification of cholesterol, and delivers cholesterol esters to liver and steriodogenic tissues for metabolism and excretion. These functions of HDL, mediated by its major protein component, apolipoprotein A-I (apoA-I), underlie the antiatherogenic role of this lipoprotein class. In several key steps of HDL metabolism, apoA-I undergoes conformational changes to adapt to changing lipid contents of the HDL, to allow the binding of apoA-II to HDL, and possibly to mediate interaction with membranes and with lecithin cholesterol acyltransferase (LCAT). A putative "hinge" region has been implicated in the conformational changes of apoA-I. The specific aims of this project are to identify the helix(es) of apoA-I that are responsible for the "hinge" functions and the key amino acids that modulate the conformational adaptability of the "hinge" domain, and to study the structural and dynamic differences between the "closed" and "open hinge" forms of apoA-I. To identify the helix(es) involved in the "hinge" functions of apoA-I, we propose to construct and express in E. coli mutants of apoA-I with each of the candidate helixes replaced with the first or last helixes of apoA-I, which bind tightly to lipid and are not mobile. After structural studies of the mutants by spectroscopic (CD and fluorescence) methods and investigation of their lipid-binding properties, the apoA-I mutants in defined RHDL particles will be examined for their "hinge" functions: particle rearrangement, apoA-II binding, interaction with bilayer membranes, and LCAT activation. The identity of the "hinge" helix(es) will be confirmed and the structural rearrangements and their dynamics will be studied by constructing Cys mutants of apoA-I as sites for specific crosslinking or fluorescent labeling. The hydrophobicity, charge distribution, and role of Pro residues in the "hinge" helix(es) will be assessed by mutagenic substitution of individual amino acids. These studies are expected to localize the "hinge" region of apoA-I and to clarify its mechanism in several important functions of apoA-I.