ApoA1 Determining HDL Subclasses and Atherosclerosis

描述(由申请人提供)：在人类和动物模型中，高密度脂蛋白已被证明具有动脉粥样硬化保护作用。人类高密度脂蛋白是异质性的，由两个主要的亚类组成，HDL2和HDL3。流行病学研究表明，HDL2比HDL3具有更强的动脉粥样硬化保护作用。小鼠和猪的高密度脂蛋白都是单相的。小鼠的高密度脂蛋白在大小和密度上接近人类的高密度脂蛋白2，而猪的高密度脂蛋白类似于高密度脂蛋白3。该方案的主要目标是建立一种以HDL2或HDL3为主要高密度脂蛋白亚类的小鼠模型，并在具有良好特征的小鼠动脉粥样硬化模型中测试HDL2和HDL3的动脉粥样硬化保护作用。在第一个特定目的中，我们将使用定点突变来使人apoA-I突变体或人/鼠和人/猪嵌合apoA-I，并确定它们在体外是否分别与成熟的人HDL2或HDL3表现出优先的结合。我们的目标是产生一种与人类载脂蛋白A-I的序列差异最小的蛋白质。根据我们的初步数据，我们最初将重点放在7/8螺旋之间的螺旋间转角。这个转角区域将被含有脯氨酸残基的人螺旋间转角或小鼠或猪的螺旋间转角所取代。我们的第二个目标是证明特定目的1的ApoAI突变体可以通过腺病毒介导的基因转移在apoA-I缺陷小鼠体内产生HDL2和HDL3。第三个具体目标将测试工程HDL2和HDL3在人类apoB转基因小鼠中预防动脉粥样硬化发展的有效性，将最好形成HDL2和HIDL3的apoA-I蛋白作为敲打基因表达。最终的特定目标将检验体内产生的突变体或工程高密度脂蛋白如何与参与高密度脂蛋白重塑和胆固醇外流的酶和受体相互作用。

DESCRIPTION (provided by applicant): HDL, both in humans and animal models, has been shown to be atheroprotective. Human HDL is heterogeneous and consists of two major subclasses, HDL2 and HDL3. Epidemiological studies suggest that HDL2 is more atheroprotective than HDL3. Mice and pigs have a monophasic HDL profile. In mice the HDL is close in size and density to human HDL2, and in pigs it is similar to HDL3. The primary goals of this proposal are to generate a mouse model in which HDL2 or HDL3 are the predominant HDL subclass and to test the atheroprotective effects of HDL2 and HDL3 in a well characterized murine atherosclerotic model. In the first specific aim we will use site-directed mutagenesis to make human apoA-I mutants or human/mouse and human/pig chimeric apoA-I and determine if they demonstrate a preferential association in vitro with mature human HDL2 or HDL3, respectively. Our goal is to generate a protein with minimum sequence differences from human apoA-I. Based on our preliminary data we will initially focus on the interhelical turn between helices 7/8. This turn region will be substituted with human interhelical turns containing proline residues or with interhelical turns from mouse or pig. Our second aim will be to demonstrate that selected apoAI mutants characterized in specific aim 1 can generate HDL2 and HDL3 in vivo in apoA-I deficient mice by adenoviral mediated gene transfer. The, third specific aim will test the efficacy of the engineered HDL2 and HDL3 in protecting against the development of atherosclerosis in human apoB transgenic mice expressing, as knockin genes, the apoA-I proteins that best form HDL2 and HIDL3. The final specific aim will examine how the mutants or the engineered HDLs generated in vivo interact with enzymes and receptors that are involved in HDL remodeling and cholesterol efflux.