Over 200 disease-causing mutations have been identified in the NPC1 gene. The most prevalent mutation, NPC1 I1061T , is predicted to lie within the cysteine-rich luminal domain and is associated with the classic juvenile-onset phenotype of Niemann-Pick type C disease. To gain insight into the molecular mechanism by which the NPC1 I1061T mutation causes disease, we examined expression of the mutant protein in human fibroblasts homozygous for the NPC1 I1061T mutation. Despite similar NPC1 mRNA levels between wild type and NPC1I1061T fibroblasts, NPC1 protein levels are decreased by 85% in NPC1 I1061T cells. Metabolic labeling studies demonstrate that unlike wild type protein, which undergoes a glycosylation pattern shift from Endo H-sensitive to Endo H-resistant species, NPC1I1061T protein remains almost exclusively Endo H-sensitive and exhibits a reduced half-life (t1 ⁄ 2 6.5 h) versus wild type Endo H-resistant species (t1 ⁄ 2 42 h). Treatment with chemical chaperones, growth at permissive temperature, or inhibition of proteasomal degradation increases NPC1I1061T protein levels, indicating that the mutant protein is likely targeted for endoplasmic reticulum-associated degradation (ERAD) due to protein misfolding. Overexpression of NPC1 I1061T in NPC1-deficient cells results in late endosomal localization of the mutant protein and complementation of the NPC mutant phenotype, likely due to a small proportion of the nascent NPC1 I1061T protein that is able to fold correctly and escape the endoplasmic reticulum quality control checkpoints. Our findings provide the first description of an endoplasmic reticulum trafficking defect as a mechanism for human NPC disease, shedding light on the mechanism by which the NPC1 I1061T mutation causes disease and suggesting novel approaches to treat NPC disease caused by the NPC1 I1061T mutation.

The Niemann-Pick type C1 (NPC1) protein is a key participant in intracellular trafficking of low density lipoprotein cholesterol, but its role in regulation of sterol homeostasis is not well understood. To characterize further the function of NPC1, we generated stable Chinese hamster ovary (CHO) cell lines overexpressing the human NPC1 protein (CHO/NPC1). NPC1 overexpression increases the rate of trafficking of low density lipoprotein cholesterol to the endoplasmic reticulum and the rate of delivery of endosomal cholesterol to the plasma membrane (PM). CHO/NPC1 cells exhibit a 1.5-fold increase in total cellular cholesterol and up to a 2.9-fold increase in PM cholesterol. This increase in PM cholesterol is closely paralleled by a 3-fold increase in de novo cholesterol synthesis. Inhibition of cholesterol synthesis results in marked redistribution of PM cholesterol to intracellular sites, suggesting an unsuspected role for NPC1 in internalization of PM cholesterol. Despite elevated total cellular cholesterol, CHO/NPC1 cells exhibit increased cholesterol synthesis, which may be attributable to both resistance to oxysterol suppression of sterol-regulated gene expression and to reduced endoplasmic reticulum cholesterol levels under basal conditions. Taken together, these studies provide important new insights into the role of NPC1 in the determination of the levels and distribution of cellular cholesterol.Intracellular cholesterol sorting and transport pathways play an important role in the physiologic utilization of lipoprotein-derived cholesterol. Low density lipoprotein (LDL) 1 and modified lipoprotein particles are trafficked to lysosomes, where the cholesteryl esters are hydrolyzed to free cholesterol (1). The bulk of LDL cholesterol is mobilized from lysosomes to the plasma membrane (PM) and subsequently cycles back to the endoplasmic reticulum (ER) (2). Approximately one-third of the unesterified lysosomal cholesterol is delivered directly to the ER via a PM-independent transport pathway (3). Cholesterol levels in the ER regulate cellular cholesterol homeostasis through a feedback regulatory mechanism that controls de novo synthesis and cellular uptake of cholesterol. This regulatory system principally involves membrane-bound transcription factors known as sterol regulatory element-binding proteins (SREBPs) (4). When cells are sterol-depleted, the NH 2 -terminal regions of the SREBPs are released through a twostep proteolytic cleavage and translocate to the nucleus to promote transcription of multiple genes involved in cholesterol and fatty acid homeostasis. When cells are replete with sterols, proteolytic cleavage of SREBPs is prevented, resulting in attenuation of SREBP-dependent gene transcription.The Niemann-Pick type C1 (NPC1) protein has been identified as a key participant in the intracellular trafficking of LDL cholesterol. Cells that harbor mutations in NPC1 accumulate cholesterol in lysosomes and exhibit delayed sterol-regulated gene expression (5). The human NPC1 gene and its murine ortholog have b...

The Niemann-Pick C1 (NPC1) protein is a key participant in intracellular sterol trafficking and regulation of cholesterol homeostasis. NPC1 contains a pentahelical region that is evolutionarily related to sterol-sensing domains found in other polytopic proteins involved in sterol interactions or sterol metabolism, including sterol regulatory element-binding protein cleavage-activating protein and hydroxymethylglutaryl-CoA reductase. To gain insight into the role of the sterol-sensing domain of NPC1, we examined the effect of point mutations in the NPC1 sterol-sensing domain on the trafficking of low density lipoprotein-derived cholesterol and sphingolipids. We show that an NPC1 P692S loss of function mutation results in decreased cholesterol delivery to the plasma membrane and endoplasmic reticulum. By contrast, NPC1 proteins carrying a L657F or D787N point mutation, which correspond to the activating SCAP L315F and D443N mutations, respectively, exhibit a gain of function phenotype. Specifically, cell lines expressing the NPC1 L657F or D787N mutations show a nearly 2-fold increase in the rates of low density lipoprotein cholesterol trafficking to the plasma membrane and to the endoplasmic reticulum, and more rapid suppression of sterol regulatory element-binding protein-dependent gene expression. Trafficking of sphingolipids is intact in the D787N and L657F cell lines. Our finding that D787N and L657F are activating NPC1 mutations provide evidence for a conserved mechanism for the sterol-sensing domain among cholesterol homeostatic proteins.