Binding and intracellular transport of 25-hydroxycholesterol by Niemann-Pick C2 protein

摘要: Abstract Side-chain oxidized cholesterol derivatives, like 25-hydroxycholesterol (25-OH-Chol) are important regulators of cellular homeostasis. How transport oxysterols through the endo-lysosomal pathway contributes to their biological function is not clear. The Niemann-Pick C2 protein (NPC2) a small lysosomal sterol transfer required for export from late endosomes and lysosomes (LE/LYSs). Here, we show that 25-hydroxy-cholestatrienol, (25-OH-CTL), an intrinsically fluorescent analogue 25-OH-Chol, becomes trapped in LE/LYSs NPC2-deficient fibroblasts, but can efflux cells even absence NPC2 upon removal source. Fluorescence recovery after photobleaching (FRAP) 25-OH-CTL endo-lysosomes was rapid extensive only partially dependent on function. Using quenching NPC2's intrinsic fluorescence, 25-OH-Chol bind though with lower affinity compared its analogues, cholestatrienol (CTL) dehydroergosterol (DHE). This confirmed by calculations binding energies which additionally two orientations NPC2, stark contrast analogues. We conclude all sterols energetically favored over self-aggregation lumen. Lysosomal strictly protein.