Regulation of Sterol Transport between Membranes and NPC2

Abstract

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.

Lipids regulate the hydrolysis of membrane bound glucosylceramide by lysosomal β-glucocerebrosidase

“…Compared with earlier experiments that used 23 mol% cholesterol in a detergentfree liposomal assay mimicking the intralysosomal situation (11), recent studies indicate that the purified lysosomes contain only traces (as low as 5 mol%) of cholesterol (13,14), much lower than the cholesterol content of the late endosomes. In the late endosomes, nondegradable cholesterol is sorted out mainly by two cholesterol transport proteins, Niemann-Pick disease protein types C1 and C2 (NPC1 and NPC2), as reported earlier (15)(16)(17)(18)(19). Of all the lipid catabolism stimulating anionic membrane lipids, BMP is the unique anionic phospholipid specifically found in lysosomes and late endosomes (6) that could reach up to 30-60 mol% of the total phospholipids in the lysosomal internal membranes (13,20).…”

Reversal of defective lysosomal transport in NPC disease ameliorates liver dysfunction and neurodegeneration in the npc1 −/− mouse

“…Originally, it seemed reasonable that during lysosomal processing of CE, the sequence of events was for LAL to release C to the soluble NPC2, which, in turn, would then transfer it to NPC1 for translocation across the limiting membrane. Recent studies, however, have raised the possibility that the direction of C movement may be from LAL to NPC1, from NPC1 to NPC2, and from NPC2 to some as yet undescribed transporter located in the limiting membrane (29,30). If this scenario is correct, CYCLO might act in the late E/L compartment by shuttling C between LAL and NPC2, thereby bypassing the defective NPC1.…”

Niemann-Pick C disease and mobilization of lysosomal cholesterol by cyclodextrin

“…Moreover, mutants of NPC2 that bind cholesterol, but cannot transfer cholesterol to NPC1, can restore cholesterol traffi cking to mitochondria in NPC2-defi cient cells ( 44 ). Because NPC2 transfers cholesterol directly to membranes ( 23 ), a possible explanation for this difference between NPC1 and NPC2 is that NPC2 transfers cholesterol from the LE/L lumen directly to the perimeter membrane of LEs/Ls, as well as to transmembrane proteins such as MLN64 and NPC1.…”

“…Fluorescence dequenching assays show that NPC2 rapidly transfers cholesterol, but not glycosphingolipids, ceramide, phospholipids, or fatty acids ( 16,19,21 ), between phospholipid liposomes in vitro. This transfer is promoted by an acidic environment and is markedly stimulated by the presence of bis(monoacylglycerol) phosphate ( 16,23 ), a phospholipid that is highly enriched in lumenal multivesicular bodies of LEs/Ls and accumulates in NPC-defi cient cells ( 24 ). X-ray crystallographic studies showed that orientation of the cholesterol molecule attached to NPC2 is opposite from that of cholesterol bound to the hydrophobic pocket of NPC1 ( 17,18,(20)(21)(22).…”