NIEMANN-PICK C AND INTRACELLULAR CHOLESTEROL TRANSPORT

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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.