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

Publication date: Available online 12 September 2019Source: Biochimica et Biophysica Acta (BBA) - BiomembranesAuthor(s): Daniel Petersen, Peter Reinholdt, Maria Szomek, Selina Kruuse Hansen, Vasanthanathan Poongavanam, Alice Dupont, Christian W. Heegaard, Kathiresan Krishnan, Hideji Fujiwara, Douglas F. Covey, Daniel S. Ory, Jacob Kongsted, Daniel WüstnerAbstractSide-chain oxidized cholesterol derivatives, like 25-hydroxycholesterol (25-OH-Chol) are important regulators of cellular cholesterol homeostasis. How transport of oxysterols through the endo-lysosomal pathway contributes to their biological function is not clear. The Niemann-Pick C2 protein (NPC2) is a small lysosomal sterol transfer protein required for export of cholesterol from late endosomes and lysosomes (LE/LYSs). Here, we show that 25-hydroxy-cholestatrienol, (25-OH-CTL), an intrinsically fluorescent analogue of 25-OH-Chol, becomes trapped in LE/LYSs of NPC2-deficient fibroblasts, but can efflux from the cells even in the absence of NPC2 upon removal of the sterol source. Fluorescence recovery after photobleaching (FRAP) of 25-OH-CTL in endo-lysosomes was rapid and extensive and only partially dependent on NPC2 function. Using quenching of NPC2's intrinsic fluorescence, we show that 25-OH-Chol and 25-OH-CTL can bind to NPC2 though with lower affinity compared to cholesterol and its fluorescent analogues, cholestatrienol (CTL) and dehydroergosterol (DHE). This is confirmed by calculations of binding energies w...
Source: Biochimica et Biophysica Acta (BBA) Biomembranes - Category: Biochemistry Source Type: research