Niemann Pick C2 protein enables cholesterol transfer from endo-lysosomes to the plasma membrane for efflux by shedding of extracellular vesicles
| Autor: | Maria Szomek, Christian W. Heegaard, Gerd Schneider, James G. McNally, Stephan Werner, Sergey Kapishnikov, Frederik W. Lund, Maria Louise V. Jensen, Alice Dupont Juhl, Peter Guttmann, Daniel Wüstner |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Niemann-Pick type C disease
Endosome 030303 biophysics Vesicular Transport Proteins ATP-binding cassette transporter Cholesterol analog Biochemistry Fluorescence Image analysis Extracellular Vesicles 03 medical and health sciences chemistry.chemical_compound Humans Cholesterol efflux Liver X receptor Molecular Biology Cells Cultured 030304 developmental biology 0303 health sciences Cholesterol Vesicle Cell Membrane Organic Chemistry Cell Biology Extracellular vesicles Soft X-ray tomography Sterol chemistry Biophysics lipids (amino acids peptides and proteins) Efflux Lysosomes |
| Zdroj: | Dupont Juhl, A, Lund, F W, Jensen, M L V, Szomek, M, Würtz Heegaard, C, Guttmann, P, Werner, S, McNally, J, Schneider, G, Kapishnikov, S & Wüstner, D 2021, ' Niemann Pick C2 protein enables cholesterol transfer from endo-lysosomes to the plasma membrane for efflux by shedding of extracellular vesicles ', Chemistry and Physics of Lipids, vol. 235, 105047 . https://doi.org/10.1016/j.chemphyslip.2020.105047 Juhl, A D, Lund, F W, Jensen, M L V, Szomek, M, Heegaard, C W, Guttmann, P, Werner, S, McNally, J, Schneider, G, Kapishnikov, S & Wüstner, D 2021, ' Niemann Pick C2 protein enables cholesterol transfer from endo-lysosomes to the plasma membrane for efflux by shedding of extracellular vesicles ', Chemistry and Physics of Lipids, vol. 235, 105047 . https://doi.org/10.1016/j.chemphyslip.2020.105047 |
| DOI: | 10.1016/j.chemphyslip.2020.105047 |
| Popis: | The Niemann-Pick C2 protein (NPC2) is a sterol transfer protein in the lumen of late endosomes and lysosomes (LE/LYSs). Absence of functional NPC2 leads to endo-lysosomal buildup of cholesterol and other lipids. How NPC2’s known capacity to transport cholesterol between model membranes is linked to its function in living cells is not known. Using quantitative live-cell imaging combined with modeling of the efflux kinetics, we show that NPC2-deficient human fibroblasts can export the cholesterol analog dehydroergosterol (DHE) from LE/LYSs. Internalized NPC2 accelerated sterol efflux extensively, accompanied by reallocation of LE/LYSs containing fluorescent NPC2 and DHE to the cell periphery. Using quantitative fluorescence loss in photobleaching of TopFluor-cholesterol (TF-Chol), we estimate a residence time for a rapidly exchanging sterol pool in LE/LYSs localized in close proximity to the plasma membrane (PM), of less than one min and observed non-vesicular sterol exchange between LE/LYSs and the PM. Excess sterol was released from the PM by shedding of cholesterol-rich vesicles. The ultrastructure of such vesicles was analyzed by combined fluorescence and cryo soft X-ray tomography (SXT), revealing that they can contain lysosomal cargo and intraluminal vesicles. Treating cells with apoprotein A1 and with nuclear receptor liver X-receptor (LXR) agonists to upregulate expression of ABC transporters enhanced cholesterol efflux from the PM, at least partly by accelerating vesicle release. We conclude that NPC2 inside LE/LYSs facilitates non-vesicular sterol exchange with the PM for subsequent sterol efflux to acceptor proteins and for shedding of sterol-rich vesicles from the cell surface. The Niemann-Pick C2 protein (NPC2) is a sterol transfer protein in the lumen of late endosomes and lysosomes (LE/LYSs). Absence of functional NPC2 leads to endo-lysosomal buildup of cholesterol and other lipids. How NPC2’s known capacity to transport cholesterol between model membranes is linked to its function in living cells is not known. Using quantitative live-cell imaging combined with modeling of the efflux kinetics, we show that NPC2-deficient human fibroblasts can export the cholesterol analog dehydroergosterol (DHE) from LE/LYSs. Internalized NPC2 accelerated sterol efflux extensively, accompanied by reallocation of LE/LYSs containing fluorescent NPC2 and DHE to the cell periphery. Using quantitative fluorescence loss in photobleaching of TopFluor-cholesterol (TF-Chol), we estimate a residence time for a rapidly exchanging sterol pool in LE/LYSs localized in close proximity to the plasma membrane (PM), of less than one min and observed non-vesicular sterol exchange between LE/LYSs and the PM. Excess sterol was released from the PM by shedding of cholesterol-rich vesicles. The ultrastructure of such vesicles was analyzed by combined fluorescence and cryo soft X-ray tomography (SXT), revealing that they can contain lysosomal cargo and intraluminal vesicles. Treating cells with apoprotein A1 and with nuclear receptor liver X-receptor (LXR) agonists to upregulate expression of ABC transporters enhanced cholesterol efflux from the PM, at least partly by accelerating vesicle release. We conclude that NPC2 inside LE/LYSs facilitates non-vesicular sterol exchange with the PM for subsequent sterol efflux to acceptor proteins and for shedding of sterol-rich vesicles from the cell surface. |
| Databáze: | OpenAIRE |
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