NIEMANN-PICK C AND INTRACELLULAR CHOLESTEROL TRANSPORT

提出的研究的总体目标是研究细胞内胆固醇运输的机制，特别是胆固醇从内核体/溶酶体排出。这些研究将利用自然实验，鼻咽癌疾病，其中至少有两个不同的缺陷基因损害胆固醇的输出，导致内体/溶酶体胆固醇积累和神经退行性表型。近年来，利用定位克隆技术分离到了18号染色体上的NPC1基因。4.5 kb的cDNA编码了一个新的1278残基多肽，该多肽具有几个假定的跨膜区域，可能参与胆固醇运输（例如转运蛋白，泵，对接蛋白等）。初步研究将确定野生型NPC1蛋白的亚细胞位置和拓扑结构。免疫组织化学和免疫电镜使用单克隆和多克隆抗体将被用来确定NPC1的亚细胞位置。五个NPC1预测的亲水环的细胞质或管腔拓扑结构将通过表达和分析一系列在每个环中带有标记标签的NPC1 cdna来评估。我们的研究结果表明，NPC1的甾醇感应结构域（SSD）与HMG-CoA还原酶和SCAP的取向相同，其拓扑结构已知。我们将进一步评估NPC1固态硬盘的意义。此外，我们的研究表明，环“c”在功能上很重要，因为在该环中含有FLAG序列的构建体不能补足NPC成纤维细胞。为了确定结构/功能关系，将表达假定的功能域，并评估其对内源性蛋白的潜在抑制作用。这些研究将增强我们对亚细胞胆固醇转运和代谢的理解，并为鼻咽癌的发病机制提供见解。我们应该强调，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（解决了该多聚糖蛋白的完整拓扑结构）方面做出了重大贡献，并确定了一个新的蛋白质靶向基序，NPC1的功能域和与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.