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• W2015964534 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 (Kd 1.4 × 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 μM 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 [14C]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. 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 (Kd 1.4 × 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 μM 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 [14C]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. Oxysterols are oxygenated derivatives of cholesterol formed either enzymatically, mainly by the action of cytochrome P450 sterol hydroxylases or through nonenzymatic cholesterol autoxidation [reviewed in (1Björkhem I. Diczfalusy U. Oxysterols: friends, foes, or just fellow passengers?.Arterioscler. Thromb. Vasc. Biol. 2002; 22: 734-742Crossref PubMed Scopus (254) Google Scholar, 2Gill S. Chow R. Brown A.J. Sterol regulators of cholesterol homeostasis and beyond: the oxysterol hypothesis revisited and revised.Prog. Lipid Res. 2008; 47: 391-404Crossref PubMed Scopus (177) Google Scholar, 3Russell D.W. Oxysterol biosynthetic enzymes.Biochim. Biophys. Acta. 2000; 1529: 126-135Crossref PubMed Scopus (303) Google Scholar–4Schroepfer Jr., G.J. Oxysterols: modulators of cholesterol metabolism and other processes.Physiol. Rev. 2000; 80: 361-554Crossref PubMed Scopus (817) Google Scholar)]. Oxysterols are present in cells at very low amounts compared with cholesterol, but they are suggested to execute important signaling functions. Certain oxysterols activate liver X receptors, key transcriptional regulators of cholesterol homeostasis (5Zelcer N. Tontonoz P. Liver X receptors as integrators of metabolic and inflammatory signaling.J. Clin. Invest. 2006; 116: 607-614Crossref PubMed Scopus (756) Google Scholar), and inhibit the processing of sterol regulatory element binding proteins (SREBPs) via binding to the Insig proteins, which retain SREBP/SCAP complexes in the endoplasmic reticulum (ER) (6Radhakrishnan A. Ikeda Y. Kwon H.J. Brown M.S. Goldstein J.L. Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: oxysterols block transport by binding to Insig.Proc. Natl. Acad. Sci. USA. 2007; 104: 6511-6518Crossref PubMed Scopus (419) Google Scholar). The cytosolic oxysterol receptor, oxysterol binding protein (OSBP), was identified in the 1980s (7Taylor F.R. Kandutsch A.A. Oxysterol binding protein.Chem. Phys. Lipids. 1985; 38: 187-194Crossref PubMed Scopus (74) Google Scholar). Families of OSBP-related proteins (ORPs) have recently been identified in practically all eukaryotic organisms studied (8Yan D. Olkkonen V.M. Characteristics of oxysterol binding proteins.Int. Rev. Cytol. 2008; 265: 253-285Crossref PubMed Scopus (46) Google Scholar). Most of the information on the ORP proteins has been obtained using yeast (Saccharomyces cerevisiae) or mammalian cells. The yeast ORPs (Osh proteins) are suggested to play major roles in the intracellular transport of sterols (9Raychaudhuri S. Im Y.J. Hurley J.H. Prinz W.A. Nonvesicular sterol movement from plasma membrane to ER requires oxysterol-binding protein-related proteins and phosphoinositides.J. Cell Biol. 2006; 173: 107-119Crossref PubMed Scopus (210) Google Scholar), in vesicle budding from the Golgi apparatus (10Fairn G.D. McMaster C.R. Emerging roles of the oxysterol-binding protein family in metabolism, transport, and signaling.Cell. Mol. Life Sci. 2008; 65: 228-236Crossref PubMed Scopus (57) Google Scholar, 11Fang M. Kearns B.G. Gedvilaite A. Kagiwada S. Kearns M. Fung M.K. Bankaitis V.A. Kes1p shares homology with human oxysterol binding protein and participates in a novel regulatory pathway for yeast Golgi-derived transport vesicle biogenesis.EMBO J. 1996; 15: 6447-6459Crossref PubMed Scopus (171) Google Scholar), and in the establishment of cell polarity (12Saito K. Fujimura-Kamada K. Hanamatsu H. Kato U. Umeda M. Kozminski K.G. Tanaka K. Transbilayer phospholipid flipping regulates Cdc42p signaling during polarized cell growth via Rga GTPase-activating proteins.Dev. Cell. 2007; 13: 743-751Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 13Schulz T.A. Prinz W.A. Sterol transport in yeast and the oxysterol binding protein homologue (OSH) family.Biochim. Biophys. Acta. 2007; 1771: 769-780Crossref PubMed Scopus (72) Google Scholar), while mammalian ORPs have been suggested to participate in the regulation of lipid metabolism, vesicle transport, and cellular signaling (8Yan D. Olkkonen V.M. Characteristics of oxysterol binding proteins.Int. Rev. Cytol. 2008; 265: 253-285Crossref PubMed Scopus (46) Google Scholar).All ORPs contain in their C-terminal part a structure designated OSBP-related domain (ORD), which is homologous to the oxysterol binding domain of OSBP (8Yan D. Olkkonen V.M. Characteristics of oxysterol binding proteins.Int. Rev. Cytol. 2008; 265: 253-285Crossref PubMed Scopus (46) Google Scholar). In addition to the ORD, most ORPs contain an N-terminal region involved in their subcellular targeting. The N-terminal extensions containing a pleckstrin homology domain target ORP1L to late endosomes (14Johansson M. Bocher V. Lehto M. Chinetti G. Kuismanen E. Ehnholm C. Staels B. Olkkonen V.M. The two variants of oxysterol binding protein-related protein-1 display different tissue expression patterns, have different intracellular localization, and are functionally distinct.Mol. Biol. Cell. 2003; 14: 903-915Crossref PubMed Scopus (89) Google Scholar), ORP3, -6, and -7 to the plasma membrane (15Lehto M. Hynynen R. Karjalainen K. Kuismanen E. Hyvärinen K. Olkkonen V.M. Targeting of OSBP-related protein 3 (ORP3) to endoplasmic reticulum and plasma membrane is controlled by multiple determinants.Exp. Cell Res. 2005; 310: 445-462Crossref PubMed Scopus (63) Google Scholar), and OSBP (16Levine T.P. Munro S. The pleckstrin homology domain of oxysterol-binding protein recognises a determinant specific to Golgi membranes.Curr. Biol. 1998; 8: 729-739Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 17Ridgway N.D. Dawson P.A. Ho Y.K. Brown M.S. Goldstein J.L. Translocation of oxysterol binding protein to Golgi apparatus triggered by ligand binding.J. Cell Biol. 1992; 116: 307-319Crossref PubMed Scopus (236) Google Scholar) and ORP9 (18Wyles J.P. Ridgway N.D. VAMP-associated protein-A regulates partitioning of oxysterol-binding protein-related protein-9 between the endoplasmic reticulum and Golgi apparatus.Exp. Cell Res. 2004; 297: 533-547Crossref PubMed Scopus (83) Google Scholar) to the Golgi complex. OSBP is targeted to Golgi complex in response to treatment of cells with its high-affinity ligand 25-hydroxycholesterol (25OHC). In several cases, the ORP pleckstrin homology domains are known to mediate the binding of phosphoinositides, but also the ORDs of ORP1, -2, -9, and -10 seem to be capable of binding these negatively charged phospholipids (19Fairn G.D. McMaster C.R. The roles of the human lipid-binding proteins ORP9S and ORP10S in vesicular transport.Biochem. Cell Biol. 2005; 83: 631-636Crossref PubMed Scopus (19) Google Scholar, 20Fairn G.D. McMaster C.R. Identification and assessment of the role of a nominal phospholipid binding region of ORP1S (oxysterol-binding-protein-related protein 1 short) in the regulation of vesicular transport.Biochem. J. 2005; 387: 889-896Crossref PubMed Scopus (29) Google Scholar–21Hynynen R. Laitinen S. Käkelä R. Tanhuanpää K. Lusa S. Ehnholm C. Somerharju P. Ikonen E. Olkkonen V.M. Overexpression of OSBP-related protein 2 (ORP2) induces changes in cellular cholesterol metabolism and enhances endocytosis.Biochem. J. 2005; 390: 273-283Crossref PubMed Scopus (68) Google Scholar). A motif called FFAT (two phenylalanines in an acidic track), which mediates the binding to the ER resident protein VAP-A (for vesicle-associated membrane protein-associated protein), specifies association of several ORPs with the ER membranes (22Loewen C.J. Roy A. Levine T.P. A conserved ER targeting motif in three families of lipid binding proteins and in Opi1p binds VAP.EMBO J. 2003; 22: 2025-2035Crossref PubMed Scopus (437) Google Scholar).Lipid droplets (LDs) are intracellular organelles composed of a neutral lipid core surrounded by a monolayer of phospholipids and cholesterol (23Fujimoto T. Ohsaki Y. Cheng J. Suzuki M. Shinohara Y. Lipid droplets: a classic organelle with new outfits.Histochem. Cell Biol. 2008; 130: 263-279Crossref PubMed Scopus (273) Google Scholar, 24Tauchi-Sato K. Ozeki S. Houjou T. Taguchi R. Fujimoto T. The surface of lipid droplets is a phospholipid monolayer with a unique fatty acid composition.J. Biol. Chem. 2002; 277: 44507-44512Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar–25Thiele C. Spandl J. Cell biology of lipid droplets.Curr. Opin. Cell Biol. 2008; 20: 378-385Crossref PubMed Scopus (225) Google Scholar). In adipocytes, their main function is to store energy in the form of triglycerides (TGs), while in steroidogenic cells, LDs store cholesteryl esters (CEs) to be used as precursors for steroid hormone biosynthesis. In other cell types, LDs serve as storage organelles for fatty acids and cholesterol needed for cell membrane renewal and signaling purposes.Whereas the role of ORPs in cholesterol metabolism has been addressed in a number of studies, their possible roles in TG metabolism have gained less interest. In the study of Yan and Olkkonen (8Yan D. Olkkonen V.M. Characteristics of oxysterol binding proteins.Int. Rev. Cytol. 2008; 265: 253-285Crossref PubMed Scopus (46) Google Scholar), overexpression of OSBP in mouse liver resulted in an increased amount of TG in liver tissue as well as enhanced secretion of TG into the circulation, apparently due to elevated activity of the lipogenic transcription factor SREBP-1c. On the other hand, overexpression of ORP2, a ubiquitously expressed ORP family member (26Laitinen S. Lehto M. Lehtonen S. Hyvarinen K. Heino S. Lehtonen E. Ehnholm C. Ikonen E. Olkkonen V.M. ORP2, a homolog of oxysterol binding protein, regulates cellular cholesterol metabolism.J. Lipid Res. 2002; 43: 245-255Abstract Full Text Full Text PDF PubMed Google Scholar), was shown to decrease the amount of both TG and CE in Chinese hamster ovary (CHO) cells (27Käkelä R. Tanhuanpää K. Laitinen S. Somerharju P. Olkkonen V.M. Overexpression of OSBP-related protein 2 (ORP2) in CHO cells induces alterations of phospholipid species composition.Biochem. Cell Biol. 2005; 83: 677-683Crossref PubMed Scopus (16) Google Scholar). In this study, we examine further the role of ORP2 in neutral lipid metabolism in the human A431 cell system. We show that ORP2 binds several oxysterols and localizes on the surface of intracellular LDs. Silencing of ORP2 affects the rate of TG hydrolysis and the degree of cholesterol esterification, thus suggesting that ORP2 acts as a sterol receptor involved in the integrated control of cholesterol and TG metabolism.EXPERIMENTAL PROCEDUREScDNA constructs, site-directed mutagenesis, short interfering RNAs, and antibodiescDNAs for transient transfections were subcloned into the pcDNA4HisMaxC expression vector (Invitrogen, Carlsbad, CA), which encodes hexahistidine and Xpress™ epitope tags fused at the N terminus of the insert proteins. ORP2 cDNA engineered by PCR to remove the stop codon was also inserted in the BamHI site of pHcRed-N1 (BD Biosciences Clontech, San Jose, CA). For protein production in Escherichia coli, cDNAs were subcloned in the BamHI site of pGEX-1λT (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The full-length ORP open reading frames used in this study were ORP1L (AF323726), ORP2 (BC000296), and ORP3 (NM_015550). The glutathione S-transferase (GST)-MLN64 START domain construct was a kind gift from Prof. Elina Ikonen (Institute of Biomedicine, University of Helsinki, Finland).The sequences of ORP2 short interfering RNA (siRNA) duplexes (Sigma Genosys, St. Louis, MO) were as follows: ORP2 siRNA 1, GGGAAGAUUUAGGAUUCAGtt (sense) and CUGAAUCCUAAAUCUUCCCtg (antisense); ORP2 siRNA 2, CGUAUGAAUUAAUCAGGGAtt (sense) and UCCCUGAUUAAUUCAUACGtt (antisense); ORP2 siRNA 3, GGACACAUUCAAGACAAAAtt (sense) and UUUUGUCUUGAAUGUGUCCtt (antisense); and ORP2 siRNA 4, GGACCGGCAAACCAUUUAAtt (sense) and UUAAAUGGUUUGCCGGUCCtc (antisense). The sequences of control siRNA duplexes were UAGCGACUAAACACAUCAATT (sense) and UUGAUGUGUUUAGUCGCUATT (antisense). The rabbit ORP2 antibody was described in (26Laitinen S. Lehto M. Lehtonen S. Hyvarinen K. Heino S. Lehtonen E. Ehnholm C. Ikonen E. Olkkonen V.M. ORP2, a homolog of oxysterol binding protein, regulates cellular cholesterol metabolism.J. Lipid Res. 2002; 43: 245-255Abstract Full Text Full Text PDF PubMed Google Scholar). A monoclonal mouse antibody (mab) against the Xpress™ epitope tag was purchased from Invitrogen, the mab against GM-130 from BD Biosciences-Transduction Laboratories (San Jose, CA), the mab against transferrin receptor from Zymed (San Francisco, CA), the mab (clone H4A3) against Lamp1 from Developmental Studies Hybridoma Bank (Iowa City, IA), and the mab against GAPDH from Novus Biologicals (Littleton, CO). Alexa-labeled fluorescent goat secondary antibody conjugates were from Invitrogen.Cell cultureThe human epithelial carcinoma cell line A431 was grown in a 5% CO2 atmosphere in DMEM with 4.5 g/l glucose and sodium pyruvate (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS (Invitrogen-Gibco BRL, Carlsbad, CA), 100 IU/ml penicillin,and 100 μg/ml streptomycin at 37°C for a maximum of 30 passages. The cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. siRNA-mediated gene silencing was carried out with HiPerFect™ (Qiagen) according to the manufacturer’s instructions using the fast-forward transfection protocol. LDs were induced in A431 cells by overnight treatment with 350 μM oleic acid-BSA complexes prepared according to (28Spector A.A. Structure and lipid binding properties of serum albumin.Methods Enzymol. 1986; 128: 320-339Crossref PubMed Scopus (113) Google Scholar). Stably transfected ORP2 HeLa TRex cells were cultured and induced with doxycyclin as previously described (21Hynynen R. Laitinen S. Käkelä R. Tanhuanpää K. Lusa S. Ehnholm C. Somerharju P. Ikonen E. Olkkonen V.M. Overexpression of OSBP-related protein 2 (ORP2) induces changes in cellular cholesterol metabolism and enhances endocytosis.Biochem. J. 2005; 390: 273-283Crossref PubMed Scopus (68) Google Scholar).Fluorescence microscopyA431 cells were fixed with 3% (w/v) paraformaldehyde in PBS for 30 min. Permeabilization of the cells and the blocking of nonspecific binding of antibodies were done simultaneously in PBS containing 0.5% BSA and 0.1% saponin (blocking buffer) for 30 min. Primary antibodies and secondary antibody conjugates were diluted in blocking buffer and incubated for 1 h at room temperature. When LDs were stained, 50 μM Bodipy 493/503 (Invitrogen-Molecular Probes, Carlsbad, CA) was added in the secondary antibody dilution. Washes after the antibody incubations were done three times with blocking buffer. Finally, the cover slips were washed three times with PBS and once with water, after which they were mounted in Mowiol 4-88 (Sigma-Aldrich) containing 2.5% 1,4-diazabicyclo[2.2.2]octane (Sigma-Aldrich). When antibody incubations were not required, cells were stained with 50 μM Bodipy 493/503 and with 4′,6-diamidino-2-phenylindole for 30 min before fixation. Images were acquired with Zeiss Meta or Leica TCS SP1 confocal microscopes.Immunoelectron microscopyFor immunoelectron microscopy, A431 cells were transfected with ORP2-pcDNA4HisMaxC and loaded with 350 μM oleic acid-BSA complexes overnight. The cells were then fixed with 4% paraformaldehyde, scraped, and pelleted in gelatin. Polyvinylpyrrolidone/sucrose-infiltrated specimens were sectioned at −110°C and picked up in methyl cellulose-sucrose. The sections were treated with 1% fish skin gelatin (Sigma-Aldrich) and 1% BSA and incubated with the ORP2 antibody (1:70) for 1 h. Secondary antibody (Protein A-10 nm gold; University of Utrecht, The Netherlands) was applied for 1 h, and sections were embedded in uranyl acetate-methyl cellulose and examined with a Jeol 1200 EX II electron microscope.Protein purificationGST fusion proteins of ORP2 and MLN64 (Metastatic lymph node 64) START (Steroidogenic acute regulatory protein-related lipid transfer) domain were produced in E. coli BL21(DE3) and purified on glutathione sepharose 4B (GE Healthcare) according to the manufacturer's instructions. Protein concentrations of the specimens were determined by the DC assay (Bio-Rad, Hercules, CA). Before oxysterol binding experiments, the protein preparations were resolved on Laemmli gels, which were stained with Coomassie blue, scanned, and analyzed using Scion Image software (http://www.scioncorp.com/). According to this, adjustment of the protein amounts added was performed to ensure that the desired concentrations of the full-length fusion proteins were reached.Charcoal-dextran oxysterol binding assayBinding of [3H] labeled 7-ketocholesterol (65 Ci/mmol; American Radiolabeled Chemicals, St. Louis, MO), 25-hydroxycholesterol (20 Ci/mmol), 22(R)hydroxycholesterol (20 Ci/mmol; American Radiolabeled Chemicals), or 27-hydroxycholesterol (45 Ci/mmol; a gift from Prof. Ingemar Bjo¨rkhem, Karolinska Institute, Huddinge, Sweden) to the purified GST-ORP2 was assayed as described previously (29Taylor F.R. Kandutsch A.A. Use of oxygenated sterols to probe the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase and sterologenesis.Methods Enzymol. 1985; 110: 9-19Crossref PubMed Scopus (15) Google Scholar). Briefly, proteins (0.25 or 1 μM) were incubated overnight at +4°C with 5, 10, 20, 40, and 80 nM (in some cases up to 160 nM) [3H]oxysterol in the absence or presence of a 40-fold excess of the corresponding unlabeled oxysterol (purchased from Sigma-Aldrich; except 27-hydroxycholesterol, which was from I. Bjo¨rkhem). The free sterol was thereafter removed with charcoal-dextran, and the protein-bound [3H]sterol remaining in the supernatant was analyzed by liquid scintillation counting. Kd values were determined by Scatchard analysis. The data were normalized for specific radioactivity of the different labeled oxysterols.Cholesterol binding assayTo be able to study the interaction of ORP2 and cholesterol in solution, cholesterol was complexed with methyl-β-cyclodextrin (mβCD; Sigma-Aldrich) according to (30Leppimäki P. Mattinen J. Slotte J.P. Sterol-induced upregulation of phosphatidylcholine synthesis in cultured fibroblasts is affected by the double-bond position in the sterol tetracyclic ring structure.Eur. J. Biochem. 2000; 267: 6385-6394Crossref PubMed Scopus (26) Google Scholar). Briefly, 1 mg of mβCD was dissolved in 1 ml of binding buffer (10 mM HEPES, pH 7.4, 50 mM KCl, and 0.02% NP-40) and added on the top of the film of cholesterol and [3H]cholesterol (44 Ci/mmol; GE-Healthcare-Amersham) in a glass tube. The mixture was then probe sonicated 3 × 5 min and microcentrifuged at full speed for 15 min. For the cholesterol binding assay, 50 μg of GST (negative control), GST-ORP2, or GST-MLN64 START (positive control) were bound to glutathione sepharose beads, which were then incubated with cholesterol-mβCD (330 ng cholesterol per assay) for 30 min at room temperature. Competition of the cholesterol binding was tested using a 40-fold molar excess of 22(R)-hydroxycholesterol [22(R)OHC]. After the binding, the beads were washed and the radioactivity of the supernatant, the washes, and the pellet was measured by liquid scintillation counting.Analysis of TG synthesis and breakdownA431 cells on six-well plates were treated with siRNAs as described above. In the case of TG synthesis assay, 350 μM oleic acid-BSA complexes containing [3H]oleic acid (5 μCi per dish; 7 Ci/mmol; GE Healthcare-Amersham) were added to the cells 40 h after the transfection. The cells were harvested at different time points in 2% NaCl. Lipids were extracted using the Bligh-Dyer protocol, and the extracts were separated by TLC using hexane/diethyl ether/acetic acid/water (65:15:1:0.25). The TLC plates were stained with iodine, the TG spots identified by comigration with a triolein standard were scraped off, and the radioactivity was measured with a liquid scintillation counter. Protein concentrations of the cell specimens were measured with the DC Protein Assay (Bio-Rad), and the results were corrected with the protein amounts. In the case of TG breakdown assay, similar [3H]oleic acid-oleic acid-BSA complexes were added to cells after 24 h of transfection. After 18 h, the cells were washed three times with PBS, and the loading/labeling medium was replaced with chase medium (DMEM, 5% delipidated serum). The cells were harvested at several time points and treated further as in the TG synthesis assay.Assay for [14C]cholesterol esterificationA431 cells on 35 mm dishes were treated with siRNAs as described above. After 6 h [14C]cholesterol (0.2 μCi/well; 58 mCi/mmol; GE Healthcare-Amersham) was added to the cells for 40 h. To analyze the distribution of the [14C]label between free cholesterol and CEs, the labeled cells were harvested in 2% NaCl and the lipids extracted using the Bligh-Dyer protocol. Aliquots of the cells were used for protein determination (see above). The neutral lipids were separated by TLC using petrolether/diethyl ether/acetic acid (60:40:1). The TLC plates were stained with iodine, the cholesterol and CE spots identified based on comigration with unlabeled standards were scraped off, and the radioactivity was measured with a liquid scintillation counter.RESULTSORP2 binds several oxysterols with different affinitiesWe recently reported that ORP2 binds 25OHC with micromolar affinity (31Suchanek M. Hynynen R. Wohlfahrt G. Lehto M. Johansson M. Saarinen H. Radzikowska A. Thiele C. Olkkonen V.M. The mammalian oxysterol-binding protein-related proteins (ORPs) bind 25-hydroxycholesterol in an evolutionarily conserved pocket.Biochem. J. 2007; 405: 473-480Crossref PubMed Scopus (118) Google Scholar). We now wanted to study the oxysterol binding properties of ORP2 more extensively. For this, we employed a charcoal-dextran pull-down assay of [3H]oxysterols with GST-tagged ORP2 produced in E. coli as previously. To determine the specific binding, we performed the assays in the absence or presence of 40-fold excess of unlabeled oxysterol, and the nonspecific binding was subtracted from the total binding (Fig. 1A). In these experiments 22(R)OHC was bound by ORP2 with the highest affinity (Kd = 1.4 × 10−8 M), followed by 7-ketocholesterol (Kd = 1.6 × 10−7 M) and 25OHC, which displayed weak but reproducible binding to the protein. However, no specific binding of 27-hydroxycholesterol was detected. We also tested the oxysterol binding properties of a mutant ORP2 (ORP2 I249W) previously shown to be defective in 25OHC binding (31Suchanek M. Hynynen R. Wohlfahrt G. Lehto M. Johansson M. Saarinen H. Radzikowska A. Thiele C. Olkkonen V.M. The mammalian oxysterol-binding protein-related proteins (ORPs) bind 25-hydroxycholesterol in an evolutionarily conserved pocket.Biochem. J. 2007; 405: 473-480Crossref PubMed Scopus (118) Google Scholar). This mutant also failed to bind 22(R)OHC (Fig. 1A). From these assays, we conclude that ORP2 binds several oxysterols with different affinities, the highest affinity ligand so far being 22(R)OHC.ORP2 binds cholesterolIn a previous study, we presented data suggesting that overexpressed ORP2 enhances the intracellular transport of cholesterol (21Hynynen R. Laitinen S. Käkelä R. Tanhuanpää K. Lusa S. Ehnholm C. Somerharju P. Ikonen E. Olkkonen V.M. Overexpression of OSBP-related protein 2 (ORP2) induces changes in cellular cholesterol metabolism and enhances endocytosis.Biochem. J. 2005; 390: 273-283Crossref PubMed Scopus (68) Google Scholar). Therefore, we wanted to study whether ORP2 might, in addition to oxysterols, bind cholesterol. Since cholesterol is markedly more hydrophobic than oxysterols, we designed a cholesterol binding assay in which [3H]cholesterol is solubilized using methyl-β-cyclodextrin and subjected to pull down with GST-tagged recombinant proteins. The START domain of the cholesterol binding protein MLN64 was used as a positive (32Tsujishita Y. Hurley J.H. Structure and lipid transport mechanism of a StAR-related domain.Nat. Struct. Biol. 2000; 7: 408-414Crossref PubMed Scopus (436) Google Scholar) and GST as a negative control. In this assay, GST-ORP2 showed significant cholesterol binding over the background signal of GST. However, the binding was modest compared with that of the GST-MLN64 START domain (Fig. 1B). The specificity of the binding was tested by adding a 40-fold excess of unlabeled 22(R)OHC in the assay. The cholesterol binding was efficiently competed by the oxysterol, suggesting that the binding is specific and occurs in the same pocket as that of the oxysterols, as shown for the homologous yeast protein Osh4p (33Im Y.J. Raychaudhuri S. Prinz W.A. Hurley J.H. Structural mechanism for sterol sensing and transport by OSBP-related proteins.Nature. 2005; 437: 154-158Crossref PubMed Scopus (335) Google Scholar).ORP2 localizes to the surface of intracellular LDsThe subcellular localization of ORP2 has so far been unclear. The data of Laitinen et al. (26Laitinen S. Lehto M. Lehtonen S. Hyvarinen K. Heino S. Lehtonen E. Ehnholm C. Ikonen E. Olkkonen V.M. ORP2, a homolog of oxysterol binding protein, regulates cellular cholesterol metabolism.J. Lipid Res. 2002; 43: 245-255Abstract Full Text Full Text PDF PubMed Google Scholar) suggested partial Golgi localization of human ORP2 in CHO cells, but when overexpressed in several human cell lines under standard culture conditions, the human ORP2 was diffusely distributed throughout the cytoplasm with no sign of Golgi association (Fig. 2A–C; data not shown). In this study, Xpress epitope-tagged ORP2 was expressed in the human epithelial carcinoma cell line A431 with abundant endogenous ORP2 expression. In the normal growth medium with 10% FBS ORP2 displayed a relatively even staining of the cytoplasmic compartment with additional small dot- or ring-like ORP2-positive structures. These structures were also found to be stained with Bodipy 493/503, a neutral lipid stain, suggesting that they may represent cytoplasmic LDs (Fig. 2A–C). When the cells were treated overnight with 350 μM oleic acid-BSA to enhance lipogenesis and LD formation, prominent ORP2 localization on Bodipy 493/503-positive spherical structures was evident (Fig. 2D–F). The spherical structures were not stained with antibodies recognizing the Golgi (GM130; Fig. 2G–I), lysosomes (Lamp1; Fig. 2J–L), or early/recycling endosomes (transferrin receptor; Fig. 2M–O). We thus concluded that ORP2 localizes on the surface of neutral LDs. The LD association was not unique for A431 cells: Overexpressed ORP2 displayed LD association also in other cell lines, such as HeLa, HepG2, and Cos7 (data not shown). ORP2 was also seen on the LD of stably transfected HeLa TRex cells induced to overexpress untagged ORP2 (21Hynynen R. Laitinen S. Käkelä R. Tanhuanpää K. Lusa S. Ehnholm C. Somerharju P. Ikonen E. Olkkonen V.M. Overexpression of OSBP-related protein 2 (ORP2) induces changes in cellular cholesterol metabolism and enhances endocytosis.Biochem. J. 2005; 390: 273-283Crossref PubMed Scopus (68) Google Scholar) (Fig. 3A–C), demonstrating that the localization is not specific for the Xpress epitope-tagged protein. Furthermore, ORP2 with the fluorescent HcRed protein fused at its C terminus displayed a similar LD localization, showing that the LD distribution is not a result from an artifactual association that could arise during the immunofluorescence staining protocol (Fig. 3D–F). This observation also demonstrates that modification of the C terminus of ORP2 does not interf" @default.
• W2015964534 created "2016-06-24" @default.
• W2015964534 creator A5011998711 @default.
• W2015964534 creator A5059590931 @default.
• W2015964534 creator A5064594852 @default.
• W2015964534 creator A5067900419 @default.
• W2015964534 creator A5075525029 @default.
• W2015964534 creator A5087036071 @default.
• W2015964534 date "2009-07-01" @default.
• W2015964534 modified "2023-10-18" @default.
• W2015964534 title "OSBP-related protein 2 is a sterol receptor on lipid droplets that regulates the metabolism of neutral lipids" @default.
• W2015964534 cites W1485380500 @default.
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