APOA-I PRIMARY SEQUENCE IN HDL ASSEMBLY AND FUNCTION

描述（改编自申请人的摘要）：几个自然 apoAI的脂质结合结构域中发生的突变与 HDL浓度降低，但矛盾的是， 早发心血管疾病 一个这样的突变是Cys替换Arg 在apoAI-M（Iano）中出现的氨基酸173处的取代。 主要 本建议的目的是建立一个动物模型， apoAI-M的抗动脉粥样硬化潜力。 单拷贝，同源 将采用重组策略用以下基因替换小鼠apoAI基因： 人apoAI或apoAI-M基因，以产生具有等同的 表达人apoAI或apoAIM表达。 基因打靶 将使用来自菌株C57 BL/6的ES细胞进行程序， 动脉粥样硬化易感株，以检查apoAI-M是否 保护替代动物免于饮食诱导的动脉粥样硬化。 的 表达人apoAI的小鼠将作为研究 apoAI-M的任何保护作用的机制。 申请人将检验以下假设。 (1)背景下 动脉粥样硬化易感的C57 BL/6小鼠的致动脉粥样硬化饮食， apoAIM基因对病变发展的保护作用比 正常人apoAI。 (2)“敲入”小鼠中apoAI/apoAI-M基因型将 概括了在人类中发现的不寻常的脂质和脂蛋白谱， 因为apoAI-M仅在杂合子携带者中观察到。 (3)载脂蛋白AI-M HDL和/或变体HDL的特定亚群显著更多 比正常apoAI更有效地刺激小鼠胆固醇流出 腹腔巨噬细胞，从而减少动脉中的脂质积聚 墙 实验的第二阶段是阐明apoAI-M是否 通过二硫键形成产生的HDL颗粒上的二聚化与 具有apoAIM的保护特性 Arg-173突变为Leu 将以与apoAI-M相同的方式引入小鼠。 apoAI的脂质结合结构域中的其他Arg-Cys取代将被取代。 生成以确定这些替换作为一个类是否具有 有保护的潜力。 这些研究提供了一种新的方法， 解决重要的人类问题， 动脉粥样硬化和其他Arg-Cys取代是否具有相同的 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.