GENETIC ANALYSIS OF LYSOSOMAL ACID LIPASE FUNCTION

溶酶体酸性脂肪酶功能的遗传分析

• 批准号: 2223948
• 负责人: RICHARD A ANDERSEN
• 金额: $ 17.32万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2223948
• 关键词: Wolman's disease; autosomal recessive trait; cholesterol ester storage disease; cholesterol esters; enzyme deficiency; hydrolase; lipase; lipid metabolism; lysosomes; molecular genetics; northern blottings; phenotype; polymerase chain reaction; site directed mutagenesis; southern blotting; structural genes; tissue /cell culture; transfection

Lysosomal acid lipase/cholesteryl ester hydrolase is crucial for the intracellular hydrolysis of cholesteryl esters and triglycerides that have been taken up via receptor mediated endocytosis of lipoprotein particles. The process is central to the supply of cholesterol to cells for growth and membrane function and in the regulation of processes that are mediated by cellular cholesterol flux. This project will characterize, at the molecular genetic level, the defects that cause a deficiency of this acid lipase activity in two human diseases, Wolman disease (WD) and cholesteryl ester storage disease (CESD), both inherited as Mendelian autosomal recessive traits, with the long term objective of understanding the biochemical and physiological activities of human lysosomal acid lipase (HLAL) in relationship to normal and aberrant states of cholesteryl ester processing and storage. The deficiencies of acid lipase activity in WD and CESD, with their dramatically different biochemical and clinical phenotypes, provide an "experiment of nature" for the testing of hypotheses concerning the control of cholesterol metabolism. The observation that CESD patients exhibit premature atherosclerosis suggests the applicability of this "experiment" to cardiovascular risk in the general population. Despite the recognized importance of HLAL in the regulation of cholesterol metabolism, difficulties with experimental manipulation of the enzyme have limited progress in understanding its control and physiological role. This project will circumvent earlier problems by exploiting the recently isolated HLAL cDNA and genomic DNA clones to test hypotheses concerning the mutations causing WD and CESD: l) Defects in expression of the structural gene for acid lipase are responsible for both WD and CESD throughout the spectrum of phenotypes. This will be done by transducing the cDNA coding for functional HLAL into mutant fibroblasts using retroviral vectors to demonstrate that this structural information is sufficient to correct the deficiency. 2) The different phenotypes arise from mutations at the HLAL structural gene locus that differentially inactivate its triglyceride hydrolase and cholesteryl esterase functions. This will involve identifying the mutations that cause WD and CESD at the nucleotide sequence level by a) characterizing the functional status of mutant HLAL genes; b) determining the structure of the coding and control regions of the normal genomic locus for comparison to the structures of the loci from the mutant cells; and c) recreating the mutated sites in vitro and expressing the constructions.

溶酶体酸性脂肪酶/胆汁胆汁酯水解酶对 胆固醇酯和具有甘油三酸酯的细胞内水解 通过受体介导的脂蛋白颗粒的内吞作用吸收。 该过程是向细胞供应胆固醇以生长的核心 和膜功能以及介导过程的调节 通过细胞胆固醇通量。 这个项目将在 分子遗传水平，导致这种酸缺乏的缺陷 两种人类疾病的脂肪酶活性，沃尔曼病（WD）和胆固醇 酯储存疾病（CESD），均被遗传为Mendelian常染色体 隐性特征，长期目标是理解 人溶酶体脂肪酶的生化和生理活性 （HLAL）与胆固醇酯的正常和异常状态的关系 处理和存储。 WD和 CESD，其生化和临床明显不同 表型为测试提供了“自然实验” 关于控制胆固醇代谢的假设。 这 观察到CESD患者表现出早产的动脉粥样硬化表明 该“实验”对心血管风险的适用性 一般人口。 尽管HLAL在调节胆固醇中的重要性很重要 代谢，实验操纵酶的困难已有 了解其控制和生理作用的进展有限。 该项目将通过利用最近的问题来解决早期的问题 分离的HLAL cDNA和基因组DNA克隆以检验有关的假设 引起WD和CESD的突变： l）酸性脂肪酶的结构基因表达缺陷为 在整个表型中负责WD和CESD。 这将通过将cDNA编码用于功能HLAL来完成 使用逆转录病毒载体突变的成纤维细胞证明这一点 结构信息足以纠正缺陷。 2）不同的表型来自HLAL结构的突变 差异使其甘油三酸酯水解酶和 胆固醇酯酶功能。这将涉及确定 在核苷酸序列水平上导致WD和CESD的突变通过a） 表征突变HLAL基因的功能状态； b）确定 正常基因组的编码和控制区域的结构 与突变细胞的基因座结构进行比较； c）在体外重新创建突变的位点并表达 构造。