Cholesterol Pathways Affected by Small Molecules That Decrease Sterol Levels in Niemann-Pick Type C Mutant Cells

Niemann-Pick type C (NPC) disease is a genetically inherited multi-lipid storage disorder with impaired efflux of cholesterol from lysosomal storage organelles.The effect of screen-selected cholesterol lowering compounds on the major sterol pathways was studied in CT60 mutant CHO cells lacking NPC1 protein. Each of the selected chemicals decreases cholesterol in the lysosomal storage organelles of NPC1 mutant cells through one or more of the following mechanisms: increased cholesterol efflux from the cell, decreased uptake of low-density lipoproteins, and/or increased levels of cholesteryl esters. Several chemicals promote efflux of cholesterol to extracellular acceptors in both non-NPC and NPC1 mutant cells. The uptake of low-density lipoprotein-derived cholesterol is inhibited by some of the studied compounds.Results herein provide the information for prioritized further studies in identifying molecular targets of the chemicals. This approach proved successful in the identification of seven chemicals as novel inhibitors of lysosomal acid lipase (Rosenbaum et al, Biochim. Biophys. Acta. 2009, 1791:1155-1165)

Interactions of Ca(2+) with sphingomyelin and dihydrosphingomyelin.

The changes induced by Ca(2+) on human lens sphingolipids, sphingomyelin (SM), and dihydrosphingomyelin were investigated by infrared spectroscopy. Ca(2+)-concentration-dependent studies of the head group region revealed that, for both sphingolipids, Ca(2+) partially dehydrates some of the phosphate groups and binds to others. Ca(2+) affects the interface of each sphingolipid differently. In SM, Ca(2+) shifts the amide I' band to frequencies lower than those in dehydrated samples of SM alone. This could be attributed to the direct binding of Ca(2+) to carbonyl groups and/or strong tightening of interlipid H-bonds to levels beyond those in dehydrated samples of SM only. In contrast, Ca(2+) induces relatively minor dehydration around the amide groups of dihydrosphingomyelin and a slight enhancement of direct lipid-lipid interactions. Temperature-dependent studies reveal that 0.2 M Ca(2+) increases the transition temperature T(m) from 31.6 +/- 1.0 degrees C to 35.7 +/- 1.1 degrees C for SM and from 45.5 +/- 1.1 degrees C to 48.2 +/- 1.0 degrees C for dihydrosphingomyelin. Binding of Ca(2+) to some phosphate groups remains above T(m). The strength of the interaction is, however, weaker. This allows for the partial rehydration of these moieties. Similarly, above T(m), Ca(2+)-lipid and/or direct inter-lipid interactions are weakened and lead to the rehydration of amide groups

Isolation and lipid characterization of cholesterol-enriched fractions in cortical and nuclear human lens fibers. Invest Ophthalmol Vis Sci 44

PURPOSE. Human lens membranes contain unusually high levels of cholesterol and sphingolipids, lipids known to segregate into liquid-ordered domains. The current study was conducted to pursue the determination and characterization of these domains in membranes of clear and cataractous human lenses. METHODS. Cortical and nuclear regions of aged clear and cataractous lenses were obtained. After lysis with Triton X-100 at 4°C and sucrose linear-density centrifugation, sedimenting and nonsedimenting fractions (when present) were collected. Phospholipids were analyzed by 31 P-nuclear magnetic resonance (NMR) and mass spectrometry. Caveolae and raft markers were tested by Western blot analysis. RESULTS. Only samples from clear lenses exhibited a nonsedimenting band. Phospholipid contents were comparable for sedimenting fractions of clear and cataractous membranes. Cholesterol to phospholipid molar ratios in light-density bands were nearly 7, three times greater than in sedimenting fractions. The portion of total cholesterol present in nonsedimenting fractions increased from 5.5% in the cortex to 14% in the nucleus. Two lysophospholipids comprising approximately 10% of all phospholipids in total membranes were undetectable in nonsedimenting fractions. Caveolin-1 was enriched in these fractions. CONCLUSIONS. Phospholipid compositional differences between lighter and heavier fractions from clear lenses were relatively minor and could not, alone, account for the substantial enrichment of cholesterol in the lighter fractions. Specific proteins, such as caveolin-1, must recruit cholesterol and induce clustering. Undetectable amounts of light-density domains in cataractous membranes suggest either disruption of these aggregates and thus the function of proteins within them, possibly relevant to lens transparency, and/or greater density of these clusters due to stronger binding of insoluble crystallins to membranes. (Invest Ophthalmol Vis Sci