APOA-I PRIMARY SEQUENCE IN HDL ASSEMBLY AND FUNCTION

描述(改编自申请者的摘要)：几个自然 载脂蛋白AI脂结合结构域的突变与 高密度脂蛋白浓度降低，但矛盾的是，与 早产儿心血管疾病。一个这样的突变是Arg的半胱氨酸 ApoAI-M(Ilano)中173位氨基酸的取代。少校 本提案的目的是建立一种动物模型，在其中进行研究 载脂蛋白AI-M的抗动脉粥样硬化作用。单一拷贝，同源 将采用重组策略将小鼠载脂蛋白AI基因替换为 人类载脂蛋白AI或载脂蛋白AI-M基因创造出具有相同功能的小鼠 人载脂蛋白AI或apoAIM的表达。基因打靶 程序将使用菌株C57BL/6的ES细胞进行，并 动脉粥样硬化易感株，以检测载脂蛋白AI-M 替代动物不受饮食诱导的动脉粥样硬化的影响。这个 表达人类载脂蛋白AI的小鼠将作为研究 载脂蛋白AI-M的保护作用机制。 申请者将检验以下假设。(1)在 对动脉粥样硬化易感的C57BL/6小鼠进行致动脉粥样硬化饮食 ApoAIM基因对病变发展的保护作用将比 正常人类载脂蛋白AI。(2)“敲入”小鼠的ApoAI/ApoAI-M基因将 概括一下在人类中发现的不同寻常的脂类和脂蛋白图谱， 因为ApoAI-M仅在杂合子携带者中观察到。(3)ApoAI-M 高密度脂蛋白和/或变异高密度脂蛋白的特定子群体明显更多 载脂蛋白AI刺激小鼠胆固醇外流的效率 腹膜巨噬细胞，从而减少脂质在动脉中的积聚 墙。实验的第二阶段是为了阐明载脂蛋白AI-M 二硫键形成对高密度脂蛋白颗粒的二聚化作用 具有apoAIM假定的保护特性。Arg-173突变为Leu 将以与ApoAI-M相同的方式被引入小鼠体内。 ApoAI脂结合区上的其他Arg-Cys取代将是 生成以确定这些替换作为一个类是否具有 具有潜在的保护性。这些研究提供了一种新的方法来 解决ApoAI-M是否能够预防的重大人类问题 动脉粥样硬化和其他Arg-Cys替代是否具有相同的作用 潜力。

DESCRIPTION (Adapted from applicant's abstract): Several naturally occurring mutations in the lipid binding domain of apoAI are associated with a decreased HDL concentration but paradoxically are not associated with premature cardiovascular disease. One such mutation is the Cys for Arg substitution at amino acid 173, which occur in apoAI-M(ilano). The major aim of the present proposal is to create an animal model in which to study the anti-atherogenic potential of apoAI-M. A single copy, homologous recombination strategy will be employed to replace the mouse apoAI gene with the human apoAI or the apoAI-M gene to create mice having equivalent expression of human apoAI or apoAIM expression. The gene targeting procedure will be carried out using ES cells from strain C57BL/6, an atherosclerosis susceptible strain, in order to examine whether the apoAI-M replacement animals are protected from dietary induced atherosclerosis. The mice expressing the human apoAI will serve as controls in studying the mechanism of any protective effect of apoAI-M. The applicant will test the following hypotheses. (1) in the background of the atherosclerosis susceptible C57BL/6 mouse on an atherogenic diet, the apoAIM gene will be more protective against lesion development than the normal human apoAI. (2) the apoAI/apoAI-M genotype in "knock-in" mice will recapitulate the unusual lipid and lipoprotein profile found in humans, since apoAI-M had only been observed in heterozygous carriers. (3) apoAI-M HDL and/or a specific sub-population of variant HDL are significantly more efficient than normal apoAI in stimulating cholesterol efflux from mouse peritoneal macrophages, thus decreasing lipid accumulation in the artery wall. The second phase of the experiments are to elucidate whether apoAI-M dimerization on HDL particles produced by disulfide bond formation is linked with apoAIM's putative protective properties. An Arg-173 to Leu mutation will be introduced into mice in the same fashion as was used for apoAI-M. Other Arg-Cys substitutions in the lipid binding domains of apoAI will be produced to determine whether these substitutions, as a class, have the potential for being protective. These studies provide a novel approach for addressing the important human issues whether apoAI-M protects against atherosclerosis and whether other Arg-Cys substitutions have the same potential.