Abstract
Oxysterol binding protein-related protein 2 (ORP2) is a member of the oxysterol binding protein family, previously shown to bind 25-hydroxycholesterol and implicated in cellular cholesterol metabolism. We show here that ORP2 also binds 22(R)-hydroxycholesterol [22(R)OHC], 7-ketocholesterol, and cholesterol, with 22(R)OHC being the highest affinity ligand of ORP2 (K(d) 1.4 x 10(-8) M). We report the localization of ORP2 on cytoplasmic lipid droplets (LDs) and its function in neutral lipid metabolism using the human A431 cell line as a model. The ORP2 LD association depends on sterol binding: Treatment with 5 microM 22(R)OHC inhibits the LD association, while a mutant defective in sterol binding is constitutively LD bound. Silencing of ORP2 using RNA interference slows down cellular triglyceride hydrolysis. Furthermore, ORP2 silencing increases the amount of [(14)C]cholesteryl esters but only under conditions in which lipogenesis and LD formation are enhanced by treatment with oleic acid. The results identify ORP2 as a sterol receptor present on LD and provide evidence for its role in the regulation of neutral lipid metabolism, possibly as a factor that integrates the cellular metabolism of triglycerides with that of cholesterol.
Highlights
Oxysterol binding protein-related protein 2 (ORP2) is a member of the oxysterol binding protein family, previously shown to bind 25-hydroxycholesterol and implicated in cellular cholesterol metabolism
We employed a charcoal-dextran pull-down assay of [3H]oxysterols with glutathione S-transferase (GST)-tagged ORP2 produced in E. coli as previously
The starting point of this study is the novel finding that ORP2 localizes on intracellular lipid droplet (LD)
Summary
Oxysterol binding protein-related protein 2 (ORP2) is a member of the oxysterol binding protein family, previously shown to bind 25-hydroxycholesterol and implicated in cellular cholesterol metabolism. When the cells were treated overnight with 350 mM oleic acid-BSA to enhance lipogenesis and LD formation, prominent ORP2 localization on Bodipy 493/503positive spherical structures was evident (Fig. 2D–F). A431 cells overexpressing ORP2 were treated overnight with 350 mM oleic acid-BSA to induce LD formation in the presence of 5 mM 22(R)OHC.
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