NIEMANN-PICK C AND INTRACELLULAR CHOLESTEROL TRANSPORT

这项研究的总体目标是研究胆固醇在细胞内的转运机制，特别是胆固醇从内小体/溶酶体中流出的机制。这些研究将利用自然界中的鼻咽癌疾病的实验，在这种疾病中，至少有两个不同的缺陷基因会损害胆固醇的输出，并导致内体/溶酶体胆固醇积聚和神经退行性表型。最近，18号染色体上的NPC1基因被定位克隆。该基因全长4.5kb，编码一个新的1278残基多肽，具有多个跨膜区，可能参与胆固醇的转运(如转运蛋白、泵、停靠蛋白等)。初步研究将确定野生型NPC1蛋白的亚细胞位置和拓扑结构。使用单抗和多克隆抗体的免疫组织化学和免疫电子显微镜将用于确定NPC1的亚细胞位置。将通过表达和分析在每个环中带有标记的一系列NPC1 cDNA来评估NPC1预测的五个亲水环的胞液或管腔拓扑。我们的结果表明，NPC1的甾醇敏感结构域(SSD)与HMG-CoA还原酶和SCAP的SSD方向相同，它们的拓扑结构是已知的。将进一步评估NPC1固态硬盘的意义。此外，我们的研究表明，环“c”具有重要的功能，因为在这个环中包含标志序列的结构不能补充鼻咽癌成纤维细胞。为了确定结构/功能关系，将表达假定的功能结构域，并评估它们对内源蛋白的潜在抑制作用。这些研究将加深我们对亚细胞胆固醇转运和代谢的理解，并为鼻咽癌疾病的发病机制提供深入的认识。需要强调的是，我们已经在NPC1的亚细胞定位(将NPC1的定位定义为晚期内体而不是溶酶体)和拓扑确定(解决了这种多位糖蛋白的完整拓扑结构)方面做出了重大贡献，并发现了一个新的靶向基序、NPC1的功能结构域和与NPC1相关的潜在85 kDa蛋白。

The overall objective of the proposed research is to investigate the mechanisms of intracellular cholesterol transport, especially cholesterol egress from the endosome/lysosome. These studies will exploit the experiment of nature, NPC disease, in which at least two different defective genes impair cholesterol egress and result in endosomal/lysosomal cholesterol accumulation and a neurodegenerative phenotype. Recently, the NPC1 gene on chromosome 18 was isolated by positional cloning. The 4.5 kb cDNA encodes a novel 1278 residue polypeptide with several putative membrane spanning regions, which presumably is involved in cholesterol transport (e.g. transporter, pump, docking protein, etc.). Initial studies will determine the subcellular location and topology o the wild-type NPC1 protein. Immunohistochemistry and immunoelectron microscopy using monoclonal and polyclonal antibodies will be used to define the subcellular location of NPC1. The cytosolic or lumenal topology of the five NPC1-predicted hydrophilic loops will be assessed by expression and analysis of a series of NPC1 cDNAs with flag-tags in each of the loops. Our results indicate that the sterol- sensing domain (SSD) of NPC1 is in the same orientation as in HMG-CoA reductase and SCAP, whose topologies are known. The significance of the NPC1 SSD will be further evaluated. In addition, our studies indicate that loop "c" is functionally significant, as constructs containing a FLAG sequence in this loop fail to complement NPC fibroblasts. To identify structure/function relationships, putative functional domains will be expressed and their potential inhibitory effects on the endogenous protein will be assessed. These studies should enhance our understanding of subcellular cholesterol transport and metabolism and provide insights into the pathogenesis of NPC disease. We should emphasize that we have already made significant contributions in the subcellular location (defined the location of NPC1 as the late endosome and not lysosomes) and topology determination of NPC1 (solved the complete topology of this polytopic glycoprotein) and have identified a novel protein targeting motif, a functional domain of NPC1 and a potential 85 kDa protein that associates with NPC1.