STRUCTURE-FUNCTION OF CATALYTIC PROTEIN IN REVERSE CHOLESTEROL TRANSPORT

LCAT：胆固醇酰基转移酶（LCAT），其合成大量 胆固醇酯和胆固醇酯转移蛋白（CETP）， 它将这些从高（HDL）转移到低（LDL）和极低（VLDL） 密度脂蛋白是人体血浆中的两种主要催化因子 胆固醇代谢，并可能在决定总胆固醇水平方面发挥重要作用。 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， 最近被鉴定为载脂蛋白D（apo D）。 这些活动 在不同的生理和病理条件下， 很大程度上是事后决定的。 定点诱变 将用于定位每个蛋白质中功能重要的残基。 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 在这个酶中形成催化三联体。 我们将检验这个假设， LCAT的脂质特异性由平均疏水指数决定 一级序列丝氨酸181周围的残基。 我们将 还测试了相邻序列在以下方面起关键作用的假设： LCAT与其高密度脂蛋白底物的结合。 最后 我们将确定N-连接的碳水化合物残基的位置， 其对LCAT催化速率具有主要影响。 在CETP中，我们将测试 假设胆固醇酯和甘油三酯结合不同 但是在一级序列的C末端的相邻位点，和 将进行缺失和点突变，以定位和定义 残留物。 我们将首先使用共价化学修饰， 然后诱变以确定该位点。 最后，我们将调查 N-连接的碳水化合物残基的位置和作用， 催化活性所需。 对于CETP抑制蛋白，我们将 确认其与载脂蛋白D的同一性，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 从HDL中取代CETP。 这些研究应用分子生物学技术 研究蛋白质的调节，这些蛋白质的活性可以对 对组织和血浆胆固醇含量的影响

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.