**2.1 Phylogenetic distribution and molecular structure of OSBP/ORP family**

As a result of differential promoter usage and splicing, there are 16 major OSBP/ORP family members. The human ORP family is divided into six subfamilies based on the gene structure and amino acid sequence homology (Fig. 1). Some of the proteins, for example ORP1 and ORP4, have both short (S) and long (L) variants.

A feature in common for all ORPs is a conserved C-terminal OSBP-related domain (ORD), which contains the highly conserved "OSBP-fingerprint (OF)" sequence EQVSHHPP (Fig. 1). Most of the human ORPs belong to the long subtype have a pleckstrin homology domain (PH domain), which bind phosphoinositides (PIPs). This interaction controls the subcellular localization of the proteins. 12 out of the 16 major mammalian OSBP/ORP family members contain a FFAT motif, which is a short sequence that binds VAMP associated proteins (VAPs), integral membrane proteins of the ER. Instead of an ER targeting FFAT motif, ORP5 and ORP8 contain a putative C-terminal transmembrane segment, which anchors these proteins in the ER. The human ORP1L have at their N-terminus ankyrin repeats, which interacts with the active GTP-bound form of Rab7 on late endosomes, and thus mediates targeting of the protein to late endosomes.

Fig. 1. Domain organization of the humam OSBP/ORP family.

Human OSBP/ORP family members are arranged into subfamilies I-VI. The color codes are: red, OSBP-related domain (ORD); yellow, OSBP-fingerprint (OF) motif; green, pleckstrin homology (PH) domain; Blue, ankirin repeats; black, VAP targeting motif (FFAT motif); Orange, transmembrane domain. L and S in the protein names indicate long and short variants, respectively.

### **2.2 OSBP**

128 Dyslipidemia - From Prevention to Treatment

OSBP negatively regulates ATP-binding cassette transporter A1 (ABCA1) protein stability (Bowden and Ridgway 2008). OSBP induces upregulation of SREBP-1c and enhances hepatic

ORP1L forms a RILP-Rab7-ORP1L complex (Johansson, Rocha et al. 2007) and is involved in both protein and lipid transport functions of the late endocytic compartments (Vihervaara,

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

ORP3 may play an important role in efficient directed membrane trafficking (Lehto, Mayranpaa et al. 2008). But the direct evidence that ORP3 functions to regulate dyslipidemia

ORP4 in an interaction with intermediate filaments inhibits an intracellular cholesterol-

ORP5 may cooperate with Niemann-Pick C1 (NPC1) to mediate the exit of cholesterol from

ORP6 is identified as one of the candidate genes that are possibly involved in the regulation

SNPs near ORP7 gene show a genome-wide significant association with low-density

ORP8 negatively regulates ABCA1 expression and macrophage cholesterol efflux (Yan, Mayranpaa et al. 2008). ORP8 has the capacity to modulate lipid homeostasis and SREBP activity, probably through an indirect mechanism required Nup62 (Zhou, Li et al. 2011). The OSBPL8-ZDHHC17 region (chr12) is detected for HDL cholesterol identified by one new

ORP9 and ORP11 are dimerized and may act as an intracellular lipid sensor or transporter

ORP10 suppresses hepatic lipogenesis and very-low-density lipoprotein production (Perttila, Merikanto et al. 2009). ORP10 is genetically associated with both TG (Perttila,

SNPs in the ORP11 gene is associated with LDL cholesterol levels, hyperglycemia /diabetes as

As a result of differential promoter usage and splicing, there are 16 major OSBP/ORP family members. The human ORP family is divided into six subfamilies based on the gene structure and amino acid sequence homology (Fig. 1). Some of the proteins, for example

A feature in common for all ORPs is a conserved C-terminal OSBP-related domain (ORD), which contains the highly conserved "OSBP-fingerprint (OF)" sequence EQVSHHPP (Fig. 1). Most of the human ORPs belong to the long subtype have a pleckstrin homology domain (PH domain), which bind phosphoinositides (PIPs). This interaction controls the subcellular localization of the proteins. 12 out of the 16 major mammalian OSBP/ORP family members contain a FFAT motif, which is a short sequence that binds VAMP associated proteins (VAPs), integral membrane proteins of the ER. Instead of an ER targeting FFAT motif, ORP5 and ORP8 contain a putative C-terminal transmembrane segment, which anchors these

Merikanto et al. 2009) and LDL cholesterol level (Koriyama, Nakagami et al. 2010).

**2.1 Phylogenetic distribution and molecular structure of OSBP/ORP family** 

well as with metabolic syndrome per se (Bouchard, Faucher et al. 2009).

triglycerides (TG) with that of cholesterol (Hynynen, Suchanek et al. 2009).

transport pathway mediated by vimentin (Wang, JeBailey et al. 2002).

lipoprotein (LDL) cholesterol (Teslovich, Musunuru et al. 2010).

SNP with genome-wide significance (Ma, Yang et al. 2010).

ORP1 and ORP4, have both short (S) and long (L) variants.

of high-density lipoprotein (HDL) cholesterol levels (North, Martin et al. 2003).

endosomes/lysosomes (Du, Kumar et al. 2011).

lipogenesis (Yan, Lehto et al. 2007).

Uronen et al. 2011).

is yet to be reported.

(Zhou, Li et al. 2010).

**Structure, Tissue distribution and Intracellular localization:** OSBP belongs to the subfamily I of OSBP/ORP homologues. OSBP has an ORD, a PH domain and an FFAT motif. OSBP shares the highest degree of similarity with ORP4 and dimerizes with ORP4. In the transfected cells, some of the OSBP was distributed diffusely in the cytoplasm, and some was bound to small vesicles near the nucleus. Upon addition of 25-hydroxycholesterol, most of the OSBP became concentrated in the Golgi apparatus (Ridgway, Dawson et al. 1992).

**Molecular functions related to dyslipidemia:** OSBP is the founding member of the OSBP/ORP family. Human OSBP was cloned in 1990 (Levanon, Hsieh et al. 1990) and was studied intensively (Ridgway, Dawson et al. 1992; Lagace, Byers et al. 1997; Ridgway, Badiani et al. 1998; Ridgway, Lagace et al. 1998; Storey, Byers et al. 1998; Lagace, Byers et al. 1999; Mohammadi, Perry et al. 2001; Levine and Munro 2002).

Yan et al. reported that adenovirus-mediated hepatic overexpression of OSBP induced a marked increase of VLDL TG. Also, the liver tissue TG were elevated in the AdOSBPinjected mice, and their TG secretion rate was increased by 70%. The messenger RNAs for enzymes of fatty acid synthesis and their transcriptional regulator, SREBP-1c, as well as the

Functions of OSBP/ORP Family Proteins and Their Relation to Dyslipidemia 131

Recently, Vihervaara et al. have shown that ORP1L silencing in macrophage foam cells inhibits the efflux of lipoprotein-derived endocytosed cholesterol to apolipoprotein A-I, providing evidence for the involvement of ORP1L in both protein and lipid transport

The multivesicular body(MVB) sorting pathway is known to be involved in many processes, including growth factor receptor down-regulation, exosome secretion, antigen presentation, the budding of enveloped viruses, and cytokinesis. Recently, Kobuna et al. have shown that knockdown of ORP1L induces the formation of enlarged MVBs in HeLa cells. They suggest that the proper cholesterol level of late endosomes/lysosomes generated by ORPs is required for normal MVB formation and MVB–mediated membrane protein degradation

**Structure, Tissue distribution and Intracellular localization:** ORP1S and ORP2 belong to the subfamily II of OSBP/ORP homologues. ORP1S and ORP2 have an ORD and an FFAT

ORP1S is expressed predominantly in skeletal muscle and heart (Johansson, Bocher et al. 2003). ORP2 is expressed ubiquitously in mammalian tissues. Highest mRNA levels of ORP2 are present in specific parts of the central nervous system (cerebellum, pituitary gland, pons, and putamen) as well as in leukocytes, placenta, and pancreas (Laitinen, Lehto et al. 2002). ORP1S has been reported to be largely cytosolic (Johansson, Bocher et al. 2003) and ORP2 localizes, in addition to a cytosolic fraction, on the surface of lipid droplets (LDs) and also

**Molecular functions related to dyslipidemia:** In the earlier study of Hynynen et al., overexpression of ORP2 induces enhancement of [14C]cholesterol efflux to all extracellular acceptors, which results in a reduction of cellular free cholesterol. They also show that ORP2

In their recent study, Hynynen et al. discover that ORP2 localizes not only cytosolic fraction but also on cytoplasmic LDs and reveal its function in neutral lipid metabolism. They show that 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 TG 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 (Hynynen, Suchanek et al. 2009). These results identify ORP2 as a sterol receptor present on LD and provide an evidence for its role in the regulation of neutral lipid metabolism, possibly as a factor that integrates the cellular metabolism of TG with that of cholesterol. By overexpressing all mammalian ORPs, Jansen et al. found that especially ORP1S and ORP2 enhanced PM-to-LD sterol transport. This reflected the stimulation of transport from the PM to the ER, rather than from the ER to LDs. Double knockdown of ORP1S and ORP2 inhibited sterol transport from the PM to the ER and LDs, suggesting a physiological role for these ORPs in the process (Jansen, Ohsaki et al. 2011). These findings suggest that ORP1S and ORP2 are essential in controlling cellular neutral lipid and cholesterol and has a strong

**Epidemiological study:** Epidemiological studies of ORP1S or ORP2 are not reported yet.

functions of the late endocytic compartments (Vihervaara, Uronen et al. 2011).

**Epidemiological study:** Epidemiological study of ORP1L is not reported yet.

(Kobuna, Inoue et al. 2010).

motif but lack PH domain.

**2.4 OSBPL1A (ORP1S) and OSBPL2 (ORP2)** 

the plasma membrane (PM) (Hynynen, Suchanek et al. 2009).

binds PtdIns(3,4,5)P(3) and enhances endocytosis.

association with the pathophysiology of dyslipidemia.

Insig-1 mRNA, were upregulated two-fold in the OSBP expressing livers. Silencing of OSBP in hepatocytes suppressed the induction of SREBP1-c by insulin and resulted in a reduction of TG synthesis. These results demonstrate that OSBP regulates hepatic TG metabolism and suggest the involvement of OSBP in the insulin signaling pathways that control hepatic lipogenesis (Yan, Lehto et al. 2007).

 Bowden et al. revealed that suppression of OSBP in Chinese hamster ovary cells by RNA interference resulted in increased ABCA1 protein expression and cholesterol efflux activity following induction with oxysterols or the synthetic LXR agonist TO901317. OSBP knockdown in J774 macrophages also increased ABCA1 expression in the presence and absence of LXR agonists. Their results demonstrate that OSBP opposes the activity of LXR by negatively regulating ABCA1 activity in the cytoplasm by sterol-binding domaindependent protein destabilization (Bowden and Ridgway 2008).

Cephalostatin 1, OSW-1, ritterazine B and schweinfurthin A are natural products that potently, and in some cases selectively, inhibit the growth of cultured human cancer cell lines. Recently, Burgett et al. have discovered that these molecules target OSBP and its closest paralog, ORP4L, and have named these natural products ORPphilins (Burgett, Poulsen et al. 2011). By uncovering the cellular targets of the ORPphilins, they have revealed that OSBP and ORP4L are involved in cancer cell survival.

They also show that ORPphilins perturb the cellular localization of OSBP and affect sphingomyelin biosynthesis. The ORPphilins are powerful probes of OSBP and ORP4L that will be useful in uncovering their cellular functions and their roles in human diseases.

**Epidemiological study:** Epidemiological study of OSBP is not reported yet.
