Analysis of a suppressor of the Niemann-Pick C phenotype

Niemann-Pick C 表型抑制子的分析

• 批准号: 6898964
• 负责人: LAURA LISCUM
• 金额: $ 33.52万
• 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/6898964
• 关键词: CHO cells; Niemann Pick disease; blood lipoprotein; blood lipoprotein transport; cholesterol; clinical research; endoplasmic reticulum; fluorescence microscopy; gene mutation; glycosphingolipids; human subject; intracellular transport; lipid transport; low density lipoprotein; lysosomes; microarray technology; molecular pathology; protein quantitation /detection; two dimensional gel electrophoresis; vesicle /vacuole

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(申请人提供)：尼曼-皮克C(NPC)是由两个遗传位点NPC1和NPC2中的一个突变引起的。我们的重点是NPC1，因为这个基因的突变导致了95%的临床病例。NPC1功能障碍最显著的后果是胆固醇的异常运动，导致胆固醇和神经鞘糖脂的溶酶体储存。NPC1促进脂类从胞内室向其他细胞膜转运的机制尚不清楚。目前，鼻咽癌尚无确切的治疗方法。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。我们已经分离出一个具有不寻常表型的鼻咽癌疾病的体细胞模型。中国仓鼠卵巢(CHO)突变体4-4-D(疾病)和4-4-S(抑制)与鼻咽癌成纤维细胞属于同一互补群，并含有相同的碱基插入，导致移码和终止。突变型4-4-D表现出典型的鼻咽癌溶酶体胆固醇储存表型，而突变型4-4-S在荧光显微镜下不显示胆固醇储存。4-4-S的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，突变的4-4-S仍然显示出缺陷的低密度脂蛋白刺激内质网(ER)中的酰辅酶A/胆固醇酰基转移酶(ACAT)，这是鼻咽癌的特征。我们的假设是，4-4-S抑制子是一种蛋白质，可以将胆固醇从内吞体内动员出来，但不能将胆固醇输送到内质网。为了验证这一假设，我们将实现以下目标。特定目的#1：鉴定抑制突变株4-4表型的基因产物(S)。特定目标#2：确定低密度脂蛋白-胆固醇在突变体4-4-S中的命运。特定目的#3：检测4-4-D和4-4-S细胞中鞘糖脂的细胞内转运。阐明负责从鼻咽癌溶酶体内清除胆固醇的细胞因子将揭示这种毁灭性的神经退行性疾病的潜在治疗靶点。