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

Daniel Petersen, Peter Reinholdt, Maria Szomek, Selina Kruuse Hansen, Vasanthanathan Poongavanam, Alice Dupont Juhl, Christian Würtz Heegaard, Kathiresan Krishnan, Hideji Fujiwara, Douglas F Covey, Daniel S Ory, Jacob Kongsted, Daniel Wüstner*

*Kontaktforfatter for dette arbejde

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Abstrakt

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.

OriginalsprogEngelsk
Artikelnummer183063
TidsskriftB B A - Biomembranes
Vol/bind1862
Udgave nummer2
Antal sider14
ISSN0005-2736
DOI
StatusUdgivet - 1. feb. 2020

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