dolichols and Niemann-Pick-Disease--Type-C

Niemann-Pick disease type C (NPC) is caused by defects in either the NPC1 or NPC2 gene and is characterized by accumulation of cholesterol and glycolipids in the late endosome/lysosome compartment. NPC2 is an intralysosomal protein that binds cholesterol in vitro. Previous studies demonstrated rapid rates of cholesterol transfer from NPC2 to model membranes [Cheruku, S. R., et al. (2006) J. Biol. Chem. 281, 31594-31604]. To model the potential role of NPC2 as a lysosomal cholesterol export protein, in this study we used fluorescence spectroscopic approaches to examine cholesterol transfer from membranes to NPC2, assessing the rate, mechanism, and regulation of this transport step. In addition, we examined the effect of NPC2 on the rate and kinetic mechanism of intermembrane sterol transport, to model the movement of cholesterol from internal lysosomal membranes to the limiting lysosomal membrane. The results support the hypothesis that NPC2 plays an important role in endo/lysosomal cholesterol trafficking by markedly accelerating the rates of cholesterol transport. Rates of sterol transfer from and between membranes were increased by as much as 2 orders of magnitude by NPC2. The transfer studies indicate that the mechanism of NPC2 action involves direct interaction of the protein with membranes. Such interactions were observed directly using FTIR spectroscopy and protein tryptophan spectral shifts. Additionally, cholesterol transfer by NPC2 was found to be greatly enhanced by the unique lysosomal phospholipid lyso-bisphosphatidic acid (LBPA), suggesting an important role for LBPA in NPC2-mediated cholesterol trafficking.

Topics: Animals; Biological Transport, Active; Carrier Proteins; Cattle; Cholesterol; Dolichols; Endosomes; Ergosterol; Fluorescent Dyes; Gangliosides; Glycoproteins; Humans; In Vitro Techniques; Intracellular Membranes; Kinetics; Lysosomes; Niemann-Pick Disease, Type C; Recombinant Proteins; Spectrometry, Fluorescence; Spectroscopy, Fourier Transform Infrared; Sterols; Tryptophan; Vesicular Transport Proteins

Fatty acid esterification of dolichol and cholesterol in Niemann-Pick type C1 mouse (Balb/c NIH npc1(-/-)) livers was investigated in response to treatment with peroxisomal proliferators. These inducers have hypolipidemic properties and influence the mevalonate pathway and the intracellular transport of the final products of this biosynthetic route. Such inducers are consequently interesting to use in a disease model with defective intracellular transport of lipids. In wild-type mice, the levels of dolichol and cholesterol found as free alcohols were not changed to any great extent upon treatment with the peroxisomal inducers dehydroepiandrosterone, clofibrate and diethylhexylphtalate. In contrast, the amounts of dolichyl esters increased whereas cholesteryl esters decreased by the same treatments. The rate of enzymatic esterification of dolichol in isolated microsomes was accordingly elevated after 5- to 7-day treatments with the efficient peroxisomal proliferators DEHP and PFOA, while the corresponding esterification of cholesterol was decreased. Upon peroxisomal induction in npc1(-/-) mice, the enzymatic dolichol esterification in vitro increased whereas the low concentration of dolichyl esters remained unchanged. The results thus demonstrate that the induction of fatty acid esterification of dolichol in vivo is impaired in npc1(-/-) mouse liver. It is therefore proposed that the intracellular lipid transport defect in npc1(-/-) mouse liver disables either dolichol and/or the fatty acid from reaching the site of esterification in vivo. This proposal was strengthened by the finding that the amount of dolichol was decreased in an isolated Golgi fraction from npc1(-/-) mice.

Topics: Acyltransferases; Animals; Cattle; Cholesterol Esters; Chromatography, High Pressure Liquid; Dolichols; Esterification; Fatty Acids; Intracellular Signaling Peptides and Proteins; Liver; Liver Extracts; Mice; Mice, Inbred BALB C; Microsomes, Liver; Mutation; Niemann-Pick C1 Protein; Niemann-Pick Disease, Type C; Peroxisome Proliferators; Proteins; Sterol O-Acyltransferase; Subcellular Fractions