OSBP-related protein 2 is a sterol receptor on lipid droplets that regulates the metabolism of neutral lipids

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 3 10 28 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 mM 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 (5), 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) (6). The cytosolic oxysterol receptor, oxysterol binding protein (OSBP), was identified in the 1980s (7). Families of OSBP-related proteins (ORPs) have recently been identified in practically all eukaryotic organisms studied (8). 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 (9), in vesicle budding from the Golgi apparatus (10, 11), and in the establishment of cell polarity (12, 13), while mammalian ORPs have been suggested to participate in the regulation of lipid metabolism, vesicle transport, and cellular signaling (8).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 (8). 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 Abbreviations: 22(R)OHC, 22(R)-hydroxycholesterol; 25OHC, 25-hydroxycholesterol; CE, cholesteryl ester; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; FCS, fetal calf serum; FFAT, two phenylalanines in an acidic tract; GST, glutathione S-transferase; LD, lipid droplet; mab, monoclonal mouse antibody; mbCD, methyl-b-cyclodextrin; ORD, oxysterol binding protein-related domain; ORP, oxysterol binding protein-related protein; OSBP, oxysterol binding protein; siRNA, short interfering RNA; SREBP, sterol regulatory element binding protein; TG, triglyceride.

Identification of OSBPL2 as a novel candidate gene for progressive nonsyndromic hearing loss by whole-exome sequencing

“…Knockdown of OSBPL2 expression with RNA interference increased the amounts of triglyceride and cholesterol ester. 22 The presence of OSBPL2 on lipid droplets was consistent with an inhibitory role in triglyceride, phospholipid, and cholesterol ester metabolism. 20,21 This evidence suggests that OSBPL2 plays a major role in the integrated control of cholesterol and triglyceride metabolism.…”

“…30,31 Tong et al 32 proved that the unifying role in all ORP homologs was the binding of PI [4]P to ORD, with additional sterol binding for certain homologs, and yeast complementation tests showed that PI [4]P binding to ORD is essential for function. A strictly conserved OSBP-fingerprint motif, "EQVSHHPP, " in the ORD region of OSBPL2 is a specific binding motif for the head of the group of the PI[4]P ligand, suggesting roles as phosphoinositide-binding proteins [20][21][22] (Figure 2e). Although OSBPL2 does not have a pleckstrin homology domain, it had been reported that the ORD region of OSBPL2 plays a role in binding anionic phospholipids analogous to yeast Osh3 and Osh4p.…”

“…As shown in Figures 2 and 3, the c.153_154delCT (p.Gln53Argfs*100) and c.583C>A (p.Leu195Met) mutations were located in the coding region of the OSBP-related domain (ORD) (residues 36-472), which was the most functional part of OSBPL2 for ligand binding. [20][21][22] The mutation of c.153_154delCT led to a frameshift in coding sequences with a stop codon (*) at position 100 in the shifted reading frame, which resulted in changes of 99 amino acids (residuals 53-151) and in-frame deletion of 329 amino acids in the ORD region. Meanwhile, the modeling and structure-based analysis of OSBPL2 showed that the c.583C>A (p.Leu195Met) mutation changes the shape of the protein in the ORD region (Figure 3b), which may alter its ability to bind ligands.…”

Hepatitis c virus and host cell lipids: An intimate connection

“…140 OSBP and its related proteins have been implicated in regulating numerous cellular processes, which play a central role in HCV life cycle, such as sphingomyelin synthesis, 141 autophagy, 142 and LD biogenesis. 143 In addition, OSBP contains a FFAT motif, conferring the ability to bind to the ER resident VAP (VAMP associated proteins) family of proteins. 140 VAPs represent another group of host cell factors whose involvement in the HCV replication cycle is well documented.…”

Functions of OSBP/ORP Family Proteins and Their Relation to Dyslipidemia

“…ORP1S and ORP2 enhance plasma membrane (PM)-to-lipid droplet (LD) sterol transport (Jansen, Ohsaki et al 2011). ORP2 presents on LD and has a functional role in the regulation of neutral lipid metabolism, possibly as a factor that integrates the cellular metabolism of triglycerides (TG) with that of cholesterol (Hynynen, Suchanek et al 2009). ORP3 may play an important role in efficient directed membrane trafficking (Lehto, Mayranpaa et al 2008).…”