Resumé
Side-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 which additionally show that 25-OH-CTL can bind in two orientations to NPC2, in stark contrast to cholesterol and its analogues. We conclude that NPC2's affinity for all sterols is energetically favored over their self-aggregation in the lysosomal lumen. Lysosomal export of 25-OH-Chol is not strictly dependent on the NPC2 protein.
| Originalsprog | Engelsk |
|---|---|
| Artikelnummer | 183063 |
| Tidsskrift | B B A - Biomembranes |
| Vol/bind | 1862 |
| Udgave nummer | 2 |
| Antal sider | 14 |
| ISSN | 0005-2736 |
| DOI | |
| Status | Udgivet - 1. feb. 2020 |
Fingeraftryk
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Binding and intracellular transport of 25-hydroxycholesterol by Niemann-Pick C2 protein. / Petersen, Daniel; Reinholdt, Peter; Szomek, Maria; Hansen, Selina Kruuse; Poongavanam, Vasanthanathan; Juhl, Alice Dupont; Heegaard, Christian Würtz; Krishnan, Kathiresan; Fujiwara, Hideji; Covey, Douglas F; Ory, Daniel S; Kongsted, Jacob; Wüstner, Daniel.
I: B B A - Biomembranes, Bind 1862, Nr. 2, 183063, 01.02.2020.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review
TY - JOUR
T1 - Binding and intracellular transport of 25-hydroxycholesterol by Niemann-Pick C2 protein
AU - Petersen, Daniel
AU - Reinholdt, Peter
AU - Szomek, Maria
AU - Hansen, Selina Kruuse
AU - Poongavanam, Vasanthanathan
AU - Juhl, Alice Dupont
AU - Heegaard, Christian Würtz
AU - Krishnan, Kathiresan
AU - Fujiwara, Hideji
AU - Covey, Douglas F
AU - Ory, Daniel S
AU - Kongsted, Jacob
AU - Wüstner, Daniel
PY - 2020/2/1
Y1 - 2020/2/1
N2 - Side-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 which additionally show that 25-OH-CTL can bind in two orientations to NPC2, in stark contrast to cholesterol and its analogues. We conclude that NPC2's affinity for all sterols is energetically favored over their self-aggregation in the lysosomal lumen. Lysosomal export of 25-OH-Chol is not strictly dependent on the NPC2 protein.
AB - Side-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 which additionally show that 25-OH-CTL can bind in two orientations to NPC2, in stark contrast to cholesterol and its analogues. We conclude that NPC2's affinity for all sterols is energetically favored over their self-aggregation in the lysosomal lumen. Lysosomal export of 25-OH-Chol is not strictly dependent on the NPC2 protein.
U2 - 10.1016/j.bbamem.2019.183063
DO - 10.1016/j.bbamem.2019.183063
M3 - Journal article
C2 - 31521631
VL - 1862
JO - B B A - Biomembranes
JF - B B A - Biomembranes
SN - 0005-2736
IS - 2
M1 - 183063
ER -