STRUCTURE-FUNCTION OF CATALYTIC PROTEIN IN REVERSE CHOLESTEROL TRANSPORT

卵磷脂：胆固醇酰基转移酶(LCAT)，合成大块 胆固醇酯和胆固醇酯转移蛋白(CETP)， 它将这些从高密度脂蛋白(高密度脂蛋白)转移到低密度脂蛋白(低密度脂蛋白)和极低密度脂蛋白(极低密度脂蛋白) 密度脂蛋白是人体血浆的两个主要催化因子 胆固醇代谢，并可能在决定总胆固醇中起主要作用 血浆中胆固醇的浓度和脂蛋白分布。 CETP活性可被循环抑制蛋白抑制， 最近鉴定为载脂蛋白D(ApoD)。这些人的活动 在不同的生理和病理条件下的因素 很大一部分是翻译后决定的。定点突变 将用于定位每个蛋白质中具有重要功能的残基。 在LCAT中，将通过识别 丝氨酸、组氨酸和天冬氨酸残基的位置可能使 在这种酶中形成催化三联体。我们将检验这一假设 LCAT的脂特异性由平均疏水指数决定 在初级序列的丝氨酸-181周围的残基。我们会 还要检验这样的假设，即相邻序列在 LCAT与其高密度脂蛋白底物的结合。终于 我们将确定N-连接碳水化合物残基的位置， 它们对LCAT的催化速率有很大的影响。在CETP中，我们将测试 胆固醇酯和甘油三酯结合的假说 而是在初级序列的C-末端的相邻位点，以及 将同时进行缺失和点突变，以本地化和定义 涉及残留物。我们将首先使用共价化学修饰和 然后用突变来定义这个位点。最后，我们将调查 N-连接碳水化合物残基的位置和作用，它们是 是催化活性所必需的。对于CETP抑制蛋白，我们将 确认其与载脂蛋白D的同源性，然后使用定点突变 (通过与相关脂质结合蛋白的序列相似性辅助) 定义负责配体结合和能力的区域 从高密度脂蛋白中取代CETP。这些研究应用了分子生物学技术。 研究对其活性有重大影响的蛋白质的调节 组织和血浆中的胆固醇含量。

Lecithin:cholesterol acyltransferase (LCAT), which synthesizes the bulk of cholesteryl esters, and cholesteryl ester transfer protein (CETP), which transfers these from high (HDL) to low (LDL) and very low (VLDL) density lipoproteins, are two major catalytic factors of human plasma cholesterol metabolism and may play a major role in determining the total concentration and lipoprotein distribution of cholesterol in plasma. CETP activity can be inhibited by a circulating inhibitory protein, recently identified as apolipoprotein D (apo D). The activity of these factors under different physiological and pathological conditions is in large part determined posttranslationally. Site-directed mutagenesis will be used to localize functionally important residues in each protein. In LCAT, the catalytic mechanism will be studied by identifying the location of the serine, histidine, and aspartate residues likely to make up a catalytic triad in this enzyme. We will test the hypothesis that the lipid specificity of LCAT is determined by the mean hydrophobic index of the residues surrounding serine-181 of the primary sequence. We will also test the hypothesis that a neighboring sequence plays a key role in the binding of LCAT to its high density lipoprotein substrate. Finally we will determine the location of the N-linked carbohydrate residues, which have a major effect of LCAT catalytic rate. In CETP, we will test the hypothesis that cholesteryl ester and triglyceride bind to distinct but neighboring sites at the C-terminal end of the primary sequence, and will make both deletions and point mutations to localize and define the residues involved. We will use first covalent chemical modification and then mutagenesis to define this site. Finally, we will investigate the location and role of the N-linked carbohydrate residues, which are required for catalytic activity. For the CETP inhibitor protein, we will confirm its identity with apo D, and then use site-directed mutagenesis (assisted by sequence similarities to related lipid-binding proteins) to define the regions responsible for ligand binding and for the ability to displace CETP from HDL. These studies apply molecular biology techniques to study the regulation of proteins whose activity can have major effects on tissue and plasma cholesterol content.