REGULATION OF CHOLESTEROL METABOLISM

描述（由申请方提供）：通过协调调节胆固醇摄取、合成、转运、降解和分泌来维持胆固醇稳态。胆固醇体内平衡的丧失导致疾病，如动脉粥样硬化和胆固醇结石。在美国和工业化国家，动脉粥样硬化及其临床后遗症是过早死亡和残疾的主要原因。我们最近发现并表征了一种新的核硫酸化氧固醇，5-胆甾烯-3b，25-二醇3-硫酸酯，其随着胆固醇向原代肝细胞线粒体的递送增加而显著增加。在这些细胞的线粒体和细胞核中发现了高浓度的氧化固醇，并且似乎能够激活编码胆固醇7 α-羟化酶（CYP 7A 1）和胆固醇转运蛋白（ABCA 1，ABCG 5，ABCG 8）的基因。我们的初步数据表明，胆固醇在线粒体中首先在25位被羟基化，然后在3位被硫酸化。然后，水溶性产物被运输出线粒体并进入细胞核。这种硫酸化的25-OH胆固醇似乎激活核受体并上调胆汁酸合成和胆固醇分泌。我们假设这种硫酸化胆固醇衍生物是一种新的胆固醇代谢途径的中间体。本申请的总体目标是表征这种新途径中的中间体，确定该途径中关键酶的细胞位置，并探索该途径在细胞胆固醇稳态中可能发挥的作用。具体目标是：1.表征和确定细胞核、线粒体和培养基中新型硫酸化氧固醇的化学结构;化学合成这种核硫酸化氧固醇; 2.阐明这种新型核硫酸化氧固醇在原代肝细胞中的代谢途径;和3.利用化学合成的核氧甾醇，探讨这种新型硫酸化氧甾醇在维持胆固醇稳态中的作用。本研究的成功完成将提供关于细胞内氧固醇调节细胞内胆固醇和脂质代谢的基础信息，这可能代表治疗高胆固醇血症和高脂血症的新治疗方法。

DESCRIPTION (provided by applicant): Cholesterol homeostasis is maintained through the coordinate regulation of cholesterol uptake, synthesis, transport, degradation, and secretion. Loss of cholesterol homeostasis causes diseases, such as atherosclerosis and cholesterol gallstones. Atherosclerosis and its clinical sequelae are a major cause of premature death and disability in the United States and in the industrialized would. We recently discovered and characterized a novel nuclear sulfated oxysterol, 5-cholesten-3b, 25-diol 3- sulphate, which increased dramatically with increased cholesterol delivery to mitochondria in primary hepatocytes. This oxysterol was found in high concentrations in the mitochondria and nuclei of these cells, and appears capable of activating the genes encoding cholesterol 7a-hydroxylase (CYP7A1), and cholesterol transporters (ABCA1, ABCG5, ABCG8). Our preliminary data indicate that cholesterol can be hydroxylated at 25-position first, and then sulfated at 3-hydroxy position in mitochondria. The water-soluble product is then transported out of the mitochondria and enters to the nucleus. This sulfated 25-OH cholesterol seems to activate nuclear receptor(s) and upregulates bile acid synthesis and cholesterol secretion. We hypothesize that this sulfated cholesterol derivative is an intermediate in a novel cholesterol metabolism pathway. The overall objective of this application is to characterize the intermediates in this novel pathway, determine the cellular location of the key enzymes in the pathway, and explore the role this pathway may play in cellular cholesterol homeostasis. the specific aims are: 1. To characterize and determine the chemical structures of the novel sulfated oxysterol(s) in nuclei, mitochondria, and culture media; to chemically synthesize this nuclear sulfated oxysterol; 2.To elucidate the metabolic pathway of this novel nuclear sulfated oxysterol in primary hepatocytes; and 3. To explore the role that this novel sulfated oxysterol plays in the maintenance of cholesterol homeostasis using the chemically synthesized nuclear oxysterol. The successful completion of this study will provide fundamental information regarding intracellular oxysterols regulate intracellular cholesterol and lipids metabolism, which may represent new therapeutic approaches to treatment of hypercholesterolemia and hyperlipidemia.