STRUCTURE/FUNCTION RELATIONSHIPS OF APOLIPOPROTEIN A (APOA-1)

早期冠状动脉粥样硬化在人类中的发生率 人口与高污染浓度的降低高度相关 密度脂蛋白及其主要载脂蛋白成分apoA-I。 描述高密度脂蛋白保护作用的一个假设是基于其 据称在一种被称为“反向胆固醇运输”的过程中起作用。这 假说认为细胞内游离胆固醇的外流 膜是由其在酯化反应中的利用驱动的 由卵磷脂：胆固醇酰基转移酶(LCAT)催化 高密度脂蛋白胆固醇酯的合成。这一过程可能会 促进胆固醇从外周组织向高密度脂蛋白外流 从而到达肝脏，在那里胆固醇被身体清除。 因此，apoA-I有两个重要的生理功能，它是一个关键 高密度脂蛋白因其独特的脂质结合而产生的结构成分 其次，它是高密度脂蛋白的结构组成部分 其独特的脂质结合能力，其次，它作为辅助因子 LCAT催化高密度脂蛋白转化为胆固醇酯 为了更清楚地了解关键的结构特征 ApoA-I决定了这个重要的 载脂蛋白，我们设计了研究来确定结构：功能 载脂蛋白A-I内的关系。这些研究可能会提供一种 载脂蛋白A-I激活磷脂底物和 为了具体确定apoA-I的哪些结构域最多 在激活LCAT中起重要作用。我们的方法将利用 猪瘟病毒apoA-I突变型蛋白的构建及鉴定 定点突变及其脂类结合的完整分析 亲和力和LCAT激活特性。载脂蛋白A-I两亲性α- 螺旋显示了22个氨基酸的最高种间保守性 外显子4中的酸性两亲性结构域已经被鉴定，并被 作为突变分析的目标。将产生变种人，这将使 用于评估两亲性螺旋界面的重要性 脂质结合和LCAT上的电荷密度和螺旋疏水矩 激活。将由研究直接检验的假设 在本应用中提出的是多个两亲性α-螺旋 参与稳定血脂：蛋白质相互作用所需的 胆固醇酯化和这些相同螺旋中的一个或多个 为界面磷脂提供合适的界面取向 LCAT。在这个模型中，特定的apoA-I两亲性螺旋与 磷脂双层并与边界脂分子相互作用 对于LCAT催化的酶。有可能两亲性多肽 将磷脂表面穿透到一定的临界深度， 增加sn-2脂肪酸羧基的可及性 通过调节酰基链的物理性质进行的酶攻击。 总而言之，这些研究将首次提供相关性 代表整个apoA-I分子的蛋白质二级结构， 与该多肽内的各个功能域结合，从而 有助于我们理解载脂蛋白A-I在调节胆固醇中的作用 动态平衡。

The incidence of premature coronary atherosclerosis in the human population is highly correlated to decreased concentrations of high density lipoprotein and its major apolipoprotein constituent, apoA-I. One hypothesis describing the protective effect of HDL is based on its purported role in a process called "reverse cholesterol transport". This hypothesis assumes that efflux of free cholesterol from cellular membranes is driven by its utilization in an esterification reaction catalyzed by lecithin:cholesterol acyltransferase (LCAT) during the synthesis of HDL cholesteryl esters. This process can potentially facilitate efflux of cholesterol from peripheral tissues to HDL and consequently to the liver where cholesterol is eliminated by the body. Thus, apoA-I has two important physiological functions, it is a key structural component of HDL as a result of its unique lipid binding capacity and secondly it acts as structural component of HDL as a result of its unique lipid binding capacity and secondly it acts as cofactor for the catalytic conversion of HDL cholesterol to cholesteryl ester by LCAT. In order to gain a clearer understanding of the key structural features of apoA-I that determine the dual functionality of this important apolipoprotein, we have designed studies to identify structure:function relationships within apoA-I. These studies will potentially provide a mechanistic view of apoA-I's activation of the phospholipid substrate and to specifically determine which structural domains of apoA-I are most important in the activation of LCAT. Our approach will utilize the construction nd characterization of mutant apoA-I proteins generated by site-directed mutagenesis and a complete analysis of their lipid binding affinity and LCAT activation properties. ApoA-I amphipathic alpha- helices showing the highest inter-species conservation of the 22 amino acid amphipathic domains within exon 4 have been identified and are targeted for mutation analysis. Mutants will be made which will allow for the assessment of the importance of amphipathic helix inter-facial charge density and helix hydrophobic moment on lipid binding and LCAT activation. The hypothesis that will be directly tested by the studies proposed in this application is that multiple amphipathic alpha-helices are involved in stabilizing lipid:protein interaction required for cholesterol esterification and that one or more of these same helices provide the proper interfacial orientation of boundary phospholipid of LCAT. In this model specific apoA-I amphipathic helices interact with the phospholipid bilayer and interact with the boundary lipid molecules for enzyme catalyses by LCAT. It is possible that amphipathic peptides penetrate the phospholipid surface to a certain critical depth and increase the accessibility of the sn-2 fatty acid carboxyl group to enzymatic attack by modulating the physical properties of the acyl chain. In summary, these studies will provide for the first time a correlation of protein secondary structure, representing the entire apoA-I molecule, to the individual functional domains within this polypeptide, thereby aiding our understanding of apoA-I's role in regulating cholesterol homeostasis.