ROLES OF CHOLESTERYL ESTER HYDROLASES IN THE LIVER

胆固醇酯水解酶在肝脏中的作用

• 批准号: 3246148
• 负责人: WILLIAM M GROGAN
• 金额: $ 12.57万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1996-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3246148
• 关键词: cholanate compound; cholesterol; enzyme activity; enzyme inhibitors; enzyme mechanism; genetic library; hormone regulation /control mechanism; isozymes; laboratory rat; liver metabolism; northern blottings; nucleic acid probes; nucleic acid sequence; phosphorylation; protein sequence; steroid metabolism; sterol esterase; western blottings

The hepatic cytosolic cholesteryl ester hydrolase (hCEH) purified in this laboratory exhibits properties of a regulatory enzyme, providing a potentially important control point for regulation of precursor pools for bile acid synthesis, biliary cholesterol secretion and membrane cholesterol. The long-term objective of this research is elucidation of the role of CEH in regulation of hepatic free cholesterol pools as they relate to cholesterol and bile acid metabolism and cholesterol homeostasis. A lambda gt11 cDNA library from cholestyramine treated rat liver will be screened with anti-hCEH and cDNA or oligonucleotide probes developed in this laboratory. Positive cDNA inserts will be cloned and sequenced to obtain complete mRNA and amino acid sequences to be compared with CEH isoenzymes from other organs. The hCEH will be further characterized with respect to pseudokinetic properties, as a function of substrate properties and various conditions and potential modulators with physiological relevance, such as bile acids, fatty acids, cholesterol, other amphiphiles, triacylglycerol, metal ions and protein constituents of cytosol. Mechanisms for regulation of hCEH will be characterized in rats, cultured hepatocytes and Hep G2 cells by measuring hCEH mass and specific activity and hCEH mRNA levels with Western and Northern blot analyses using anti-hCEH and a nick translated cDNA probe, under various physiological conditions or pharmacological treatments known to alter cholesterol or bile acid metabolism. Levels of protein phosphorylation will be measured where enzyme specific activities are altered and activity will be correlated with phosphorylation of specific sites. Effects of manipulation of hCEH and perturbation of free cholesterol on other key enzymes of cholesterol metabolism (acyl-CoA:cholesterol acyltransferase, HMG-CoA reductase, cholesterol 7alpha-hydroxylase), on bile acid synthesis, biliary cholesterol secretion and hepatic free and esterified cholesterol will be measured with a view to manipulation of free cholesterol pools and measurement of effects on these various activities. CEH inhibitory proteins will be isolated, sequenced and characterized in order to determine their mechanism of action and evaluate their potential physiological role in regulation of CEH. These studies will fill a significant gap in understanding of cholesterol metabolism and homeostasis and may identify a site for intervention in derangements of cholesterol/bile acid metabolism or to alter membrane properties.

在此纯化的肝胞质胆固醇酯水解酶（HCEH） 实验室展示了调节酶的特性，提供了 对前体池调节的潜在重要控制点 胆汁酸合成，胆固醇分泌和膜 胆固醇。 这项研究的长期目标是阐明 CEH在调节肝的无胆固醇池中的作用 与胆固醇和胆汁酸代谢以及胆固醇有关 稳态。 lambda gt11 cDNA库，来自胆碱治疗的大鼠 肝脏将用抗HCEH​​，cDNA或寡核苷酸探针筛选 在这个实验室中开发。 阳性cDNA插入物将被克隆，并且 测序获得完整的mRNA和氨基酸序列进行比较 来自其他器官的CEH同工酶。 HCEH将会进一步 在伪动力特性方面的特征是 底物特性以及各种条件以及潜在调节剂 生理相关性，例如胆汁酸，脂肪酸，胆固醇， 其他两亲物，三酰基甘油，金属离子和蛋白质成分 细胞质。 将在以HCEH调节的机制中进行表征 大鼠，培养的肝细胞和Hep G2细胞通过测量HCEH质量和 具有西部和北印迹的特定活性和HCEH mRNA水平 在各种 已知改变的生理状况或药理治疗 胆固醇或胆汁酸代谢。 蛋白质磷酸化水平 将在更改酶特定活动的情况下测量 活性将与特定位点的磷酸化相关。 HCEH操纵和自由胆固醇扰动的影响 胆固醇代谢的其他关键酶（酰基-COA：胆固醇 酰基转移酶，HMG-COA还原酶，胆固醇7alpha-Hydroxylase），ON） 胆汁酸合成，胆汁胆固醇分泌和无肝的胆固醇和 酯化的胆固醇将测量以操纵 游离胆固醇池和对这些各种影响的影响的测量 活动。 CEH抑制蛋白将被分离，测序，并 为了确定其作用机理的特征和 评估其在CEH调节中的潜在生理作用。 这些 研究将填补了解胆固醇的显着空白 代谢和稳态，可能会确定一个干预的地点 胆固醇/胆汁酸代谢或改变膜的危险 特性。