ApoA1 Determining HDL Subclasses and Atherosclerosis

描述（由申请人提供）：HDL在人类和动物模型中均显示出抗动脉粥样硬化作用。人HDL是异质的，并且由两个主要亚类HDL 2和HDL 3组成。流行病学研究表明，HDL 2比HDL 3更具有抗动脉粥样硬化作用。小鼠和猪具有双相HDL谱。在小鼠中，HDL在大小和密度上接近于人HDL 2，在猪中，它类似于HDL 3。该建议的主要目标是产生其中HDL 2或HDL 3是主要HDL亚类的小鼠模型，并在充分表征的小鼠动脉粥样硬化模型中测试HDL 2和HDL 3的动脉粥样硬化保护作用。在第一个具体目标中，我们将使用定点诱变来制备人apoA-I突变体或人/小鼠和人/猪嵌合apoA-I，并确定它们是否在体外分别与成熟人HDL 2或HDL 3表现出优先缔合。我们的目标是产生一种与人apoA-I序列差异最小的蛋白质。基于我们的初步数据，我们将首先关注螺旋7/8之间的螺旋间转弯。该转角区域将被含有脯氨酸残基的人螺旋间转角或来自小鼠或猪的螺旋间转角取代。我们的第二个目的是证明在特定目的1中表征的所选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HDL 2和HDL 3。第三个具体目的是测试工程化HDL 2和HDL 3在人apoB转基因小鼠中保护免于动脉粥样硬化发展的功效，所述转基因小鼠表达作为敲入基因的apoA-I蛋白，所述apoA-I蛋白最佳地形成HDL 2和HIDL 3。最后的具体目标将检查体内产生的突变体或工程化HDL如何与参与HDL重塑和胆固醇流出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.