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

人早发冠状动脉粥样硬化的发病率 人口数量与高浓度 密度脂蛋白及其主要载脂蛋白成分apoA-I。 描述HDL保护作用的一种假设是基于其 据称在一个称为“胆固醇逆向转运”的过程中起作用。 这 这一假说假定游离胆固醇从细胞中流出， 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， HDL胆固醇酯的合成。 这个过程可能会 促进胆固醇从外周组织流出到HDL， 从而进入肝脏，在那里胆固醇被身体排出。 因此，apoA-I具有两个重要的生理功能，它是一个关键 由于其独特的脂质结合， 其次，它作为HDL的结构成分， 其独特的脂质结合能力，其次，它作为辅因子， LCAT催化HDL胆固醇转化为胆固醇酯。 为了更清楚地了解关键结构特征， apoA-I决定了这个重要的 载脂蛋白，我们设计的研究，以确定结构：功能 apoA-I之间的关系 这些研究可能会提供一个 apoA-I激活磷脂底物的机制， 为了明确apoA-I的哪些结构域最重要， 在激活LCAT中起重要作用。 我们的方法将利用 构建和表征通过以下方法产生的突变apoA-I蛋白： 定点突变和它们的脂质结合的完整分析 亲和力和LCAT活化性质。 ApoA-I两亲性α- 螺旋显示22个氨基酸的最高种间保守性 外显子4内的酸性两亲性结构域已被鉴定， 用于突变分析。 突变体将被制造出来， 用于评估两亲性螺旋界面的重要性 电荷密度和螺旋疏水矩对脂质结合和LCAT的影响 activation. 这些研究将直接验证的假设是， 本申请中提出的是多个两亲性α-螺旋 参与稳定脂质：蛋白质相互作用， 胆固醇酯化和一个或多个这些相同的螺旋 提供适当的界面取向的边界磷脂 LCAT。 在该模型中，特定的apoA-I两亲性螺旋与 磷脂双层并与边界脂质分子相互作用 用LCAT进行酶催化。 两亲性肽有可能 渗透磷脂表面至某一临界深度， 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酶攻击。 总之，这些研究将首次提供一种相关性， 蛋白质二级结构，代表整个apoA-I分子， 与该多肽内的各个功能结构域连接，从而 帮助我们理解apoA-I在调节胆固醇中的作用， 体内平衡

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.