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Contents 1 Oxysterol-binding proteins 1.1 OSBP - the founder member of the family 1.2 Mammalian OSBP homologues 1.3 Yeast S. cerevisiae ORP (Osh, OSBP homologue) proteins 1.3.1 Osh4p and Golgi secretory function 1.3.2 Yeast Osh proteins act as sterol sensors/transporters? 1.4 References

Oxysterol-binding proteins

Families of proteins with homology to the carboxy-terminal ligand binding domain (denoted ORD, OSBP-related domain) of oxysterol binding protein (OSBP) are present in eukaryotic organisms from S. cerevisiae to man, and are suggested to function in various cellular processes such as lipid metabolism, intracellular lipid transport, membrane trafficking, and cell signaling (Fairn and McMaster 2008; Yan and Olkkonen 2008). In humans as well as in mice, the gene family consists of 12 members (Fig. 1), which are denoted OSBP-like genes (OSBPL), while the encoded proteins are called either OSBP-like (OSBPL) or OSBP-related proteins (ORP)(Lehto et al. 2001; Jaworski et al. 2001; Anniss et al. 2002). The fact that baker’s yeast S. cerevisiae has seven ORP genes (Beh et al. 2001) suggests a fundamental role of these genes and their products in the physiology of eukaryotic cells. In accordance with this, each human tissue or cell type expresses a large number of ORPs (Lehto et al. 2001).

Fig. 1. The human ORP family. The roman numerals indicate subfamilies of closely related proteins. OS and the orange arrows indicate documented oxysterol binding. The color codes are: Red, OSBP-related (ligand binding) domain (ORD) ; Dark blue, pleckstrin homology domain (PHD); Orange, ankyrin repeats (ANK); Pink, OSBP dimerization domain (D); Turqoise, sequence unrelated to ORD in ORP3 splice variant 2; Green, trans-membrane domain (TM). L and S in the protein names indicate long and short variants, respectively. 

OSBP - the founder member of the family

OSBP is the most extensively studied member of the mammalian ORP protein family. It was purified in the 1980s based on its ability to bind 25-hydroxycholesterol and several other oxysterols at high (nM range) affinity (Taylor et al. 1984). A shift of OSBP to a Golgi location upon treatment of cells with 25-hydroxycholesterol (Ridgway et al. 1992) coincides with Golgi translocation and activation of ceramide transport protein, CERT, a process which is inhibited when OSBP is silenced by RNA interference (Perry and Ridgway 2006). The prevailing hypothesis arising from this observation is that OSBP acts as a sterol sensor whose function is to integrate, via regulation of CERT function, the cellular sterol status with sphingomyelin metabolism. A recent report suggests that OSBP regulates in concert with the integral endoplasmic reticulum anchor protein VAP (VAMP-associated protein), CERT, and the PtdInositol/PtdCholine transfer protein Nir2, the lipid composition of the Golgi complex, impacting, in addition to sphingomyelin, also the levels of diacylglycerol and PdtIns-4-P in Golgi membranes (Peretti et al. 2008). OSBP plays, on the other hand, an important role as a sterol ligand-dependent scaffold that regulates signaling events: It is involved in the control of the phosphorylation status and activity of extracellular signal regulated kinases (Wang et al. 2005) as well as of JAK2/STAT3 activity (Romeo and Kazlauskas 2008). Interestingly, OSBP overexpression in mouse liver impacts serum very-low-density lipoprotein triglyceride (TG) levels and hepatic TG synthesis through up-regulation of the expression and activity of sterol regulatory element binding protein 1c (SREBP-1c)(Yan et al. 2007a), a major insulin-responsive regulator of hepatic and adipose tissue lipogenesis. OSBP was also shown to be required for efficient mediation of an insulin signal to the lipogenic machinery in cultured mouse hepatocytes. Since P-ERK has been reported to destabilize the active nuclear form of SREBP-1c (Botolin et al. 2006), this finding may be related to the function of OSBP as a regulator of ERK (see above).

Mammalian OSBP homologues

The mammalian OSBP-related proteins, ORPs, have typically wide tissue expression patterns, and there is considerable splice variation of their mRNAs. On the other hand, there are marked quantitative differences in their tissue- and cell-type specific expression patterns.

Three ORPs have been described as being relatively enriched in human macrophages. These are ORP1L (long splice variant), ORP3 (also abundant in T-cells, B-cells, monocytes and in certain epithelia), and ORP8.Overexpression of human ORP1L in mouse macrophages resulted in impaired cholesterol efflux and increased size of atherosclerotic lesions in LDL-receptor deficient mice (Yan et al. 2007b). ORP1L localizes to late endosomes/lysosomes and forms a complex with the small GTPase Rab7 and its other effector protein RILP (Johansson et al. 2005, 2007). Together with these partners, ORP1L controls the microtubule-dependent positioning of late endocytic compartments and may modulate vesicle transport in the late endocytic pathway. How this function relates to the increased atherosclerosis in the above in vivo experiments is unclear, However, one can envision that disturbances in late endocytic protein/lipid transport by excess ORP1L is involved. The OSBP homologue ORP8 was recently characterized as a factor that is abundant in CD68+ macrophages of human coronary artery lesions and suppresses, most likely via an indirect mechanism, the expression of ATP-binding cassette transporter A1 in THP-1 macrophages (ABCA1)(Yan et al. 2008). The third family member abundant in macrophages, ORP3, was recently reported to interact with R-Ras and to regulate cell adhesion, organization of the actin cytoskeleton, and macrophage phagocytic activity (Lehto et al. 2008).

Over-expression of ORP2 or ORP4S has been reported to impact on cellular sterol, neutral lipid or phospholipid metabolism in cultured cell models (Laitinen et al. 2002; Hynynen et al. 2005; Käkelä et al. 2005; Wang et al. 2002), lending further support to the view that ORP family members play important roles in the control of cellular lipid metabolism. Of the mammalian ORPs, ORP1S, ORP1L, ORP2, and ORP4 (also named OSBP2), have been shown to bind oxysterols (25-hydroxycholesterol, 7-ketocholesterol, 22R-hydroxycholesterol) as their ligands (Fairn and McMaster 2005a; Suchanek et al. 2007; Wang et al. 2002; Moreira et al. 2001). In addition, ORP8 was demonstrated to show some affinity for 25-hydroxycholesterol (Yan et al. 2008). Most mammalian ORPs carry a pleckstrin homology domain in the N-terminal half, with affinity for phosphoinositides; This part of the protein appears to specify subcellular localization of the proteins (Johansson et al. 2003; Lehto et al. 2005; Lagace et al. 1997; Levine and Munro 1998,2002). In addition to sterol ligands, also the ORDs of ORP1, ORP2, ORP9 and ORP10 have been suggested to show affinity for phosphoinositides (PIPs; Fairn and McMaster 2005a,2005b; Hynynen et al. 2005). The PIP interactions of the ligand binding domains are, based on data from yeast S. cerevisiae, suggested to play a role in the mechanism by which the proteins extract sterol ligands from biological membranes (Im et al. 2005).

In addition to the suggested roles of OSBP in cell signaling, also ORP9 has been implicated as a regulator of signaling events, with impact on the phosphorylation of Akt/PKB (Lessman et al. 2007).

Yeast S. cerevisiae ORP (Osh, OSBP homologue) proteins

S. cerevisiae has 7 Osh proteins, of which 3 (Osh1p-Osh3p) belong to the long category, having N-terminal extensions with a pleckstrin homology domain, and 4 (Osh4p-Osh7p) are of the short subtype consisting of not much more than a ligand binding domain (ORD). Beh et al. (2001) determined the phenotypic effects of all 127 permutations of OSH deletion alleles. The results demonstrated that the individual OSH genes are not essential, but deletion of all seven is lethal, suggesting that the genes have a shared function essential for viability. The viable combinations of OSH deletions displayed distinct sterol-related defects, and depletion of all seven proteins resulted in cellular sterol accumulation, evidencing for a disturbance of sterol homeostatic control. Elimination of OSH function resulted in ergosterol redistribution from the plasma membrane to intracellular locations, vacuolar fragmentation, and cellular accumulation of lipid droplets (Beh and Rine, 2004). Furthermore, the authors reported disturbances of endocytosis, cell budding, and cell wall deposition in cells with OSH defects.

Osh4p and Golgi secretory function

Fang et al. (1996) demonstrated that deletion of OSH4/KES1 leads to by-pass of the temperature-sensitivity of mutants in SEC14, a gene encoding a phosphatidylinositol transfer protein (PITP; Sec14p) essential for secretory vesicle biogenesis. This suggested that yeast Osh4p acts as a negative regulator of Golgi secretory function. Fairn et al. (2007) reported evidence that Osh4p impacts the synthesis of PtdIns-4-P in Golgi membranes and its availability for other proteins binding this phosphoinositide, providing one possible explanation for the involvement of this protein in Golgi secretory function.

Yeast Osh proteins act as sterol sensors/transporters?

The first ORP high-resolution structure, that of a short yeast S. cerevisiae ORP, Osh4p/Kes1p, revealed that Osh4p is a sterol-binding protein (Im et al. 2005). It was crystallized in complex with five different sterols, and has a sterol binding pocket formed by 19 beta-strands in an antiparallel arrangement. The sheet bends to an almost complete roll that is, in the presence of bound ligand, closed by a lid containing an amphipathic aplha-helix connected by a flexible linker. Sterols bind within the pocket oriented with the 3beta-hydroxyl group at the bottom of the hydrophobic binding tunnel. The sterol side chain interacts with the lid, stabilizing its closed conformation. Many of the interactions of the bound sterol are mediated via water molecules within the pocket, giving the ligand interaction substantial flexibility. This provides a plausible explanation to the ability of the pocket to accommodate structurally different sterols, and possibly also other types of lipid ligands. The structure of Osh4p suggested that this protein and its homologues might act as sterol transporters or mediators of sterol signals (Im et al. 2005). Human ORP2 structure was modeled using the Osh4p structure as template (Fig. 2). Mutagenesis based on the structural model suggests that also the human ORP2 (and possibly other ORPs) has sterol-binding pocket similar to that of Osh4p (Suchanek et al. 2007).

Fig. 2. A homology model of human ORP2 ligand binding domain structure (created using Osh4p as template). Beta-strands are shown in yellow and alpha-helices in red. A bound sterol is shown in white in the middle. (Courtesy of Dr. Gerd Wohlfahrt)

Raychaudhuri et al. (2006) presented evidence for function of the Osh proteins (Osh4p, Osh5p, and Osh3p) in sterol transport from the yeast plasma membrane to the esterification compartment, the ER. Consistent with the in vivo findings, the authors showed that Osh4p is capable of extracting sterols from donor liposomes and transferring them to acceptor vesicles in vitro. The sterol transfer was more rapid between membranes that contain PIPs, suggesting that interaction of ORPs with the negatively charged PIP headgroups on membrane surfaces facilitates the sterol transport function. Consistently, the authors found that the transport of sterol from the plasma membrane to the ER is slowed down in mutant strains with defects in PtdIns-4-P or PtdIns-4,5-P2 synthesis. It is unclear whether the yeast ORPs investigated by Raychaudhuri et al. (2006) act as true sterol transporters. According to an alternative hypothesis, the ORPs affect sterol transport indirectly by affecting the ability of the cellular plasma membrane to sequester these lipids (Sullivan et al. 2006).

References

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