Analysis of a suppressor of the Niemann-Pick C phenotype

描述（由申请人提供）：尼曼-匹克C型（NPC）是由两个遗传基因座NPC 1和NPC 2中的一个突变引起的。我们的重点是NPC 1，因为该位点的突变导致了95%的临床病例。NPC 1功能障碍的最显著后果是异常的胆固醇运动，其导致胆固醇和鞘糖脂的溶酶体储存。NPC 1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，NPC尚无明确的治疗方法。阐明抑制NPC表型的细胞因子可能揭示新的治疗靶点。 我们已经分离出一个具有异常表型的NPC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）属于与NPC成纤维细胞相同的互补组，并在NPC 1基因中包含相同的碱基插入，这导致移码和终止。突变体4-4-D显示溶酶体胆固醇储存的典型NPC疾病表型;然而，突变体4-4-S通过菲律宾荧光显微镜显示没有胆固醇储存。4-4-S表型可能是由于抑制突变表型的基因的表达。令人惊讶的是，突变体4-4-S仍然显示出有缺陷的低密度脂蛋白刺激内质网（ER）中的酰基辅酶A/胆固醇酰基转移酶（ACAT），这是NPC的特征。 我们的假设是，4-4-S抑制因子是一种将胆固醇从内体中动员出来的蛋白质，但不能将胆固醇运送到ER。为了验证这个假设，我们将执行以下目标。 具体目的#1：鉴定抑制突变体4-4的表型的基因产物。 具体目的#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。

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.