Analysis of a suppressor of the Niemann-Pick C phenotype

Niemann-Pick C 表型抑制子的分析

• 批准号: 7191648
• 负责人: LAURA LISCUM
• 金额: $ 31.78万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7191648
• 关键词: Abbreviations; BODIPY; Cell Line; Cell membrane; Cell model; Cells; Cellular Membrane; Characteristics; Chinese Hamster; Cholesterol; Class; Clinical; Cyclodextrins; D Cells; Destinations; Disease; Employee Strikes; Endoplasmic Reticulum; Endosomes; Fibroblasts; Filipin; Fluorescence Microscopy; Functional disorder; G(M3) Ganglioside; Gene Expression; Genes; Genetic; Glycosphingolipids; Goals; Green Fluorescent Proteins; Hepatosplenomegaly; Hereditary Disease; Human; LDL Cholesterol Lipoproteins; Lactosylceramides; Lead; Lipids; Lipoproteins; Low-Density Lipoproteins; Lysosomes; Mediating; Membrane; Microarray Analysis; Movement; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Newborn Infant; Ovary; Pathway interactions; Pattern; Phenotype; Plasma; Proteins; Proteome; Serum; Somatic Cell; Sphingolipids; Sterol O-Acyltransferase; Structure of thyroid parafollicular cell; Supraoptic Vertical Ophthalmoplegia; Testing; Two-Dimensional Gel Electrophoresis; Vesicle; Work; base; dichlorodifluoromethane; disease phenotype; gel electrophoresis; innovation; late endosome; lipid metabolism; lipid transport; mutant; novel therapeutics; research study; therapeutic target; trafficking; two-dimensional

DESCRIPTION (provided by applicant): Niemann-Pick C (NPC) is caused by mutations in one of two genetic loci, NPC1 and NPC2. Our focus is on NPC1 because mutations in this locus are responsible for 95% of the clinical cases. The most striking consequence of NPC1 dysfunction is aberrant cholesterol movement, which results in lysosomal storage of cholesterol and glycosphingolipids. The mechanism by which NPC1 facilitates lipid transport from endocytic compartments to other cellular membranes is unknown. Currently, there is no definitive therapy for NPC. Elucidation of cellular factors that suppress the NPC phenotype may reveal new therapeutic targets. We have isolated a somatic cell model of NPC disease with an unusual phenotype. Chinese hamster ovary (CHO) mutants 4-4-D (disease) and 4-4-S (suppressed) belong to the same complementation group as NPC fibroblasts and contain the identical base insertion in the NPC1 gene, which results in a frameshift and termination. Mutant 4-4-D shows the classical NPC disease phenotype of lysosomal cholesterol storage; however, mutant 4-4-S shows no cholesterol storage by filipin fluorescence microscopy. The 4-4-S phenotype is likely due to expression of a gene that suppresses the mutant phenotype. Surprisingly, mutant 4-4-S still shows defective low-density lipoprotein stimulation of acyl-CoA/cholesterol acyltransferase (ACAT) in the endoplasmic reticulum (ER), which is characteristic of NPC. Our hypothesis is that the 4-4-S suppressor is a protein that mobilizes cholesterol out of endosomes, but fails to deliver the cholesterol to the ER. To test this hypothesis, we will perform the following Aims. Specific Aim #1: To identify the gene product(s) that suppresses the phenotype of mutant 4-4. Specific Aim #2: To determine the fate of LDL-cholesterol in mutant 4-4-S. Specific Aim #3: To examine intracellular trafficking of glycosphingolipids in 4-4-D and 4-4-S cells. Elucidation of cellular factors responsible for cholesterol clearance from NPC lysosomes will reveal potential therapeutic targets for this devastating neurodegenerative disease.

描述（由申请人提供）：Niemann-Pick C (NPC) 是由两个基因位点 NPC1 和 NPC2 之一的突变引起的。我们的重点是 NPC1，因为该位点的突变导致了 95% 的临床病例。 NPC1 功能障碍最显着的后果是胆固醇运动异常，导致胆固醇和鞘糖脂在溶酶体中储存。 NPC1 促进脂质从内吞区室转运到其他细胞膜的机制尚不清楚。目前，鼻咽癌尚无确定的治疗方法。阐明抑制鼻咽癌表型的细胞因子可能会揭示新的治疗靶点。 我们分离出具有不寻常表型的鼻咽癌体细胞模型。中国仓鼠卵巢 (CHO) 突变体 4-4-D（疾病）和 4-4-S（抑制）与 NPC 成纤维细胞属于同一互补组，并且在 NPC1 基因中包含相同的碱基插入，从而导致移码和终止。突变体4-4-D显示溶酶体胆固醇储存的经典NPC疾病表型；然而，菲律宾荧光显微镜显示突变体 4-4-S 没有胆固醇储存。 4-4-S 表型可能是由于抑制突变表型的基因表达所致。令人惊讶的是，突变体4-4-S仍然表现出内质网（ER）中酰基辅酶A/胆固醇酰基转移酶（ACAT）的低密度脂蛋白刺激缺陷，这是NPC的特征。 我们的假设是，4-4-S 抑制剂是一种将胆固醇从内体中调动出来的蛋白质，但无法将胆固醇输送到内质网。为了检验这个假设，我们将实现以下目标。 具体目标#1：鉴定抑制突变体 4-4 表型的基因产物。 具体目标#2：确定突变体 4-4-S 中 LDL-胆固醇的命运。 具体目标#3：检查 4-4-D 和 4-4-S 细胞中鞘糖脂的细胞内运输。 阐明负责从 NPC 溶酶体清除胆固醇的细胞因子将揭示这种破坏性神经退行性疾病的潜在治疗靶点。