MUTANTS IN INTRACELLULAR CHOLESTEROL TRANSPORT

细胞内胆固醇运输的突变体

• 批准号: 2150372
• 负责人: LAURA LISCUM
• 金额: $ 21.99万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2150372
• 关键词: CHO cells; acyltransferase; cell fusion; cholesterol; complementary DNA; enzyme activity; esterification; gene complementation; gene expression; genetic library; genotype; homeostasis; human subject; intracellular transport; low density lipoprotein; mutant; northern blottings; oxidoreductase; phenotype; plasmids; polymerase chain reaction; protein transport; steroid metabolism; suppressor mutations; transfection /expression vector

Mammalian cells tightly regulate their cholesterol content and its intracellular disposition. Cholesterol is not uniformly distributed among cell membranes, and rates of cholesterol biosynthesis, lipoprotein internalization, and cholesterol esterification are sensitive to cellular levels of free cholesterol. Neither the sensing mechanism nor the mechanism by which cholesterol is compartmentalized within cells are well understood. Our long term goal is to identify gene products involved in intracellular cholesterol transport and regulation. We have isolated a battery of recessive somatic cell mutants that are defective in the intracellular transport of low density lipoprotein (LDL)-derived cholesterol. Complementation analysis reveals that at least two genes control LDL- cholesterol signaling and transport. Preliminary analysis suggests the following: Mutations in the first gene impair LDL-cholesterol egress from lysosomes. These Class l mutants appear to be a somatic cell model for classical Niemann-Pick disease type C (NPC). Mutation in the second gene impairs LDL-cholesterol signaling but not transport. These Class 2 mutants appear to be a model for a variant phenotype of NPC. We propose: Specific Aim #1: To analyze the biochemical phenotype of Class l and Class 2 complementation groups. We will thoroughly investigate how these two gene defects alter key aspects of intracellular cholesterol transport and cellular cholesterol metabolism. Specific Aim #2: To identify other Class 2 mutants and additional complementation groups. Our conclusions are based on a partial analysis of the entire collection of mutants. Fusion of CHO mutants with NPC fibroblasts will be performed to identify the mutant line with the NPC genotype. Specific Aim #3: To isolate cDNAs that correct or suppress the mutations in Class l and Class 2 mutants. Identification of cpNAs yielding normal phenotypes will reveal information on the genes that are defective in these cell lines. Specific Aim #4: To isolate and analyze CHO lines expressing dominant defects in intracellular cholesterol transport due to overexpression of a cDNA encoding a normal cellular protein. Identification of cDNAs yielding mutant phenotypes will reveal novel gene products that control cellular cholesterol distribution.

哺乳动物细胞严格调节其胆固醇含量及其 细胞内处置。胆固醇在人体内分布不均匀 细胞膜、胆固醇生物合成速率、脂蛋白 内化和胆固醇酯化对细胞敏感 游离胆固醇水平。无论是传感机制还是 胆固醇在细胞内划分的机制已明确 明白了。 我们的长期目标是识别细胞内参与的基因产物 胆固醇运输和调节。我们隔离了一个电池 隐性体细胞突变体，细胞内有缺陷 低密度脂蛋白（LDL）衍生的胆固醇的运输。 互补分析表明至少有两个基因控制 LDL- 胆固醇信号传导和运输。初步分析表明 以下：第一个基因的突变会损害低密度脂蛋白胆固醇的排出 溶酶体。这些l类突变体似乎是体细胞模型 经典尼曼-匹克病 C 型 (NPC)。第二个基因突变 损害 LDL 胆固醇信号传导，但不损害转运。这些2类突变体 似乎是 NPC 变异表型的模型。我们建议： 具体目标#1：分析 I 类和 Class 的生化表型 2个互补组。我们将彻底调查这两个 基因缺陷改变细胞内胆固醇运输的关键方面 细胞胆固醇代谢。 具体目标#2：识别其他 2 类突变体和其他突变体 互补组。我们的结论是基于部分分析 整个突变体集合。 CHO突变体与NPC的融合 将进行成纤维细胞鉴定以鉴定与 NPC 的突变系 基因型。 具体目标#3：分离纠正或抑制突变的 cDNA 在l类和2类突变体中。鉴定产生正常的 cpNA 表型将揭示有缺陷的基因的信息 这些细胞系。 具体目标#4：分离和分析表达显性基因的 CHO 系 由于a的过度表达导致细胞内胆固醇转运缺陷 编码正常细胞蛋白质的cDNA。鉴定 cDNA 产量 突变表型将揭示控制细胞的新基因产物 胆固醇分布。