NIEMANN-PICK C AND INTRACELLULAR CHOLESTEROL TRANSPORT

该研究的总体目标是研究细胞内胆固醇转运的机制，特别是胆固醇从内体/溶酶体中排出的机制。 这些研究将利用自然实验，NPC疾病，其中至少两种不同的缺陷基因损害胆固醇排出，并导致内体/溶酶体胆固醇积累和神经退行性表型。 最近，通过定位克隆分离了位于18号染色体上的NPC 1基因。 4.5kb的cDNA编码一种新的1278个残基的多肽，具有几个假定的跨膜区域，推测其参与胆固醇转运（例如转运蛋白、泵、对接蛋白等）。 初步研究将确定野生型NPC 1蛋白的亚细胞位置和拓扑结构。使用单克隆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NPC 1的亚细胞位置。 将通过表达和分析一系列在每个环中具有标记标签的NPC 1 cDNA来评估五个NPC 1预测的亲水环的胞质或内腔拓扑结构。 我们的研究结果表明，NPC 1的甾醇敏感结构域（SSD）与HMG-CoA还原酶和SCAP中的拓扑结构相同。 NPC 1 SSD的意义将进一步评估。 此外，我们的研究表明环“c”在功能上是重要的，因为在该环中含有FLAG序列的构建体不能补充NPC成纤维细胞。 为了确定结构/功能关系，将表达推定的功能结构域，并评估其对内源性蛋白的潜在抑制作用。这些研究将有助于加深我们对亚细胞胆固醇转运和代谢的理解，并为NPC疾病的发病机制提供新的见解。 我们应该强调的是，我们已经在亚细胞定位（将NPC 1的位置定义为晚期内体而不是溶酶体）和NPC 1的拓扑结构确定（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，并确定了一种新的蛋白质靶向基序，NPC 1的功能结构域和一种与NPC 1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.