Analysis of a suppressor of the Niemann-Pick C phenotype

描述（由申请人提供）：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，因为该基因座的突变导致95%的临床病例。NPC1功能障碍最显著的后果是胆固醇异常运动，导致溶酶体储存胆固醇和鞘糖脂。NPC1促进脂质从内吞室转运到其他细胞膜的机制尚不清楚。目前，对于鼻咽癌没有明确的治疗方法。抑制鼻咽癌表型的细胞因子的阐明可能揭示新的治疗靶点。我们已经分离出一个具有不寻常表型的鼻咽癌的体细胞模型。中国仓鼠卵巢（CHO）突变体4-4-D（患病）和4-4-S（抑制）与NPC成纤维细胞属于同一互补群，在NPC1基因中含有相同的碱基插入，导致移位和终止。突变体4-4-D表现为典型的NPC病溶酶体胆固醇储存表型；然而，突变体4-4-S在filipin荧光显微镜下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表达所致。令人惊讶的是，突变型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抑制因子是一种将胆固醇从核内体中动员出来的蛋白质，但不能将胆固醇输送到内质网。为了验证这一假设，我们将执行以下目的。具体目标1：确定抑制突变体4-4表型的基因产物。具体目标2：确定突变型4-4-S中ldl -胆固醇的命运。特异性目的#3：检测4-4-D和4-4-S细胞内鞘糖脂的运输。阐明鼻咽癌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.