期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 275, 期 17, 页码 12769-12780出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.275.17.12769
关键词
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资金
- NCRR NIH HHS [5P41RR03155] Funding Source: Medline
- NIDDK NIH HHS [DK41463] Funding Source: Medline
- NIGMS NIH HHS [GM 31561] Funding Source: Medline
Fluorescent sterols, dehydroergosterol and NBD-cholesterol, were used to examine high density lipoprotein-mediated cholesterol uptake and intracellular targeting in L-cell fibroblasts. The uptake, but not esterification or targeting to lipid droplets, of these sterols differed >100-fold, suggesting significant differences in uptake pathways. NBD cholesterol uptake kinetics and lipoprotein specificity reflected high density lipoprotein-mediated sterol uptake via the scavenger receptor B1. Fluorescence energy transfer showed an average intermolecular distance of 26 (A) over dot between the two fluorescent sterols in L-cells. Indirect immunofluorescence revealed that both fluorescent sterols localized to L-cell lipid droplets, the surface of which contained adipose differentiation-related protein. This lipid droplet-specific protein specifically bound NBD-cholesterol with high affinity (K-d = 2 nM) at a single site. Thus, NBD-cholesterol and dehydroergosterol were useful fluorescent probes of sterol uptake and intracellular sterol targeting. NBD-cholesterol more selectively probed high density lipoprotein-mediated uptake and rapid intracellular targeting of sterol to lipid droplets. Targeting of sterol to lipid droplets was correlated with the presence of adipose differentiation related protein, a lipid droplet-specific protein shown for the first time to bind unesterified sterol with high affinity.
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