Biochem. J. (2010) 429, 13–24 (Printed in Great Britain)
13
doi:10.1042/BJ20100263
REVIEW ARTICLE
Functional implications of sterol transport by the oxysterol-binding protein
gene family
Mike H. NGO, Terry R. COLBOURNE and Neale D. RIDGWAY1
Department of Pediatrics, Biochemistry and Molecular Biology, Atlantic Research Centre, Dalhousie University, Halifax, Nova Scotia, Canada
Cholesterol and its numerous oxygenated derivatives (oxysterols)
profoundly affect the biophysical properties of membranes, and
positively and negatively regulate sterol homoeostasis through
interaction with effector proteins. As the bulk of cellular sterols are
segregated from the sensory machinery that controls homoeostatic
responses, an important regulatory step involves sterol transport or
signalling between membrane compartments. Evidence for rapid,
energy-independent transport between organelles has implicated
transport proteins, such as the eukaryotic family of OSBP
(oxysterol-binding protein)/ORPs (OSBP-related proteins). Since
the founding member of this family was identified more than
25 years ago, accumulated evidence has implicated OSBP/ORPs
in sterol signalling and/or sterol transport functions. However,
recent evidence of sterol transfer activity by OSBP/ORPs suggests
that other seemingly disparate functions could be the result of
alterations in membrane sterol distribution or ancillary to this
primary activity.
INTRODUCTION
almost 0.1 % of the entire human genome [8,9]. Members of
these protein families have verified cholesterol/oxysterol-binding
motifs adjacent to domains with regulatory and membranetargeting functions. The present review will focus on the
OSBP/ORP family in the context of integration of sterol binding
and transport functions with effects on signalling, vesicular
trafficking and lipid metabolism. Where necessary, structural and
functional parallels with yeast OSHs (OSBP homologues) will be
identified (but see also the recent comprehensive reviews on this
subject [10,11]).
The intracellular pathways for cholesterol synthesis, uptake
and efflux are subject to negative and positive feedback
by direct interaction of cholesterol or its oxygenated
derivatives (oxysterols) with regulatory components, including
the SREBP (sterol-regulatory-element-binding protein)–SREBPcleavage-activating protein–Insig complex [1,2], HMG-CoA (3hydroxy-3-methylglutaryl-CoA) reductase [3] and LXRs (liver
X receptors) [4,5]. These reside in relatively cholesterol-poor
compartments that are subject to acute changes in local cholesterol
and oxysterol content. Although there is substantial information
on sterol interaction with this regulatory machinery, the proximal
events that control delivery of cholesterol and oxysterols to
relevant compartments are poorly understood. On the basis
of evidence that differentiates vesicular from protein-mediated
cholesterol transport, it is proposed that the latter accounts for
the majority of cholesterol delivery between organelles [6,7].
However, identification of soluble cholesterol/oxysterol transport
proteins has been problematic due to functional redundancy,
inadequate probes or end-points to monitor cholesterol movement
in vivo and difficulties in the identification of organelle-specific
targeting mechanisms. Recent interest has focused on START
[StAR (steroidogenic acute regulatory) protein-related lipid
transfer] and the OSBP (oxysterol-binding protein)/ORP (OSBPrelated protein) families that are encoded by 27 genes comprising
Key words: cholesterol, oxysterol, oxysterolbinding protein
(OSBP), oxysterol-binding protein-related protein (ORP),
pleckstrin homology (PH) domain, sterol transport, vesicleassociated membrane protein-associated protein (VAP).
PHYLOGENETIC DISTRIBUTION AND GENE EXPRESSION
Data mining of EST (expressed sequence tag), cDNA and genomic
databases revealed that the human OSBP family consists of OSBP
and ORP1–11 (also termed OSBP1, OSBP2 and OSBP-like 1–3
and 5–11) [12–14]. Mammalian ORPs are grouped into six subfamilies based on sequence identity (Figure 1): subfamily I, OSBP
and ORP4; subfamily II, ORP1 and 2; subfamily III, ORP3, 6 and
7; subfamily IV, ORP5 and 8; subfamily V, ORP9; and subfamily
VI, ORP10 and 11 [13]. The defining feature of this gene family is
a C-terminal sterol-binding or OH (OSBP homology) domain that
harbours a signature sequence (EQVSHHPP) that is conserved in
orthologues from other species. With the exception of ORP2, fulllength or L (‘long’) gene products have N-terminal PH (pleckstin
homology) domains that interact with PIPs (phosphoinositide
Abbreviations used: ABC, ATP-binding cassette; ApoA1, apolipoprotein A-I; APP, amyloid precursor protein; Arf, ADP-ribosylation factor; Aβ, amyloid
β-peptide; BRAM, BMP (bone morphogenic protein) receptor-associated protein; CERT, ceramide transfer protein; CHO, Chinese-hamster ovary;
CK1, casein kinase 1; ER, endoplasmic reticulum; ERK, extracellular-signal-regulated kinase; FFAT, two phenylalanines (FF) in an acidic tract; HCV,
hepatitis C virus; HEK, human embryonic kidney; HePTP, haematopoietic tyrosine phosphatase; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; (22R )-OH,
(22R )-hydroxycholesterol; 25-OH, 25-hydroxycholesterol; LDL, low-density lipoprotein; LDLR, LDL receptor; LE, late endosome; LXR, liver X receptor;
miRNA, microRNA; NS5A, non-structural protein 5A; NVJ, nuclear–vacuolar junction; OH domain, OSBP homology domain; ORP, OSBP-related protein;
OSBP, oxysterol-binding protein; OSBP-Dm, D. melanogaster OSBP homologue; OSH, OSBP homologue; oxLDL, oxidized LDL; PDK, phosphoinositidedependent kinase; PH, pleckstrin homology; PiORP1, Petunia inflata oxysterol binding-protein-related protein 1; PIP, phosphoinositide phosphate; PKD,
protein kinase D; PM, plasma membrane; PP2, protein phosphatase 2; RNAi, RNA interference; SM, sphingomyelin; SNP, single nucleotide polymorphism;
SREBP, sterol-regulatory-element-binding protein; START, StAR (steroidogenic acute regulatory) protein-related lipid transfer; TAG, triacylglycerol; TGF-β,
transforming growth factor-β; TGN, trans -Golgi network; VAP, VAMP (vesicle-associated membrane protein)-associated protein; VLDL, very-low-density
lipoprotein.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2010 Biochemical Society
14
Figure 1
M. H. Ngo, T. R. Colbourne and N. D. Ridgway
Structural organization of the human OSBP/ORP family
Human OSBP/ORP family members are arranged into subfamilies I–VI. Alternate nomenclature
is indicated in parentheses next to each OSBP/ORP. The Figure only includes the truncated or
S (‘short’) variants where the protein expression was verified in cell lines or tissue. Individual
domains are colour-coded: black, ankyrin; green, PH; blue, FFAT; red, OH (sterol-binding); and
yellow, transmembrane. The hatched line at the N-terminus of ORP4S indicates two potential
translation start sites.
phosphates). All OSBP/ORPs, with the exception of subfamily
IV and VI, also contain a FFAT motif [two phenylalanines (FF)
in an acidic tract] that binds VAP [VAMP (vesicle-associated
membrane protein)-associated protein] on the ER (endoplasmic
reticulum). This function seems to be fulfilled in subfamily IV
by a unique C-terminal transmembrane domain that mediates ER
localization. The functional significance of these domains will be
elaborated below; however; the initial impression is of a family
of sterol-binding proteins capable of interaction with the ER and
other organelle membranes.
ORP genes also give rise to truncated or S (‘short’) variants
with unique expression and functional properties as a result
of alternative promoter and splice-site usage. For example, an
alternate promoter prior to exon 3 in the ORP4 gene gives rise to
a short (ORP4S) variant missing the PH domain [15] (Figure 1).
Alternative promoters before exons 16 and 6 of the ORP1 and
ORP9 genes respectively result in expression of ORP1S
and ORP9S [16,17]. As many as eight ORP3 isoforms are
produced as a result of alternative splicing of exons 9, 12 and
15 [18].
OSBP orthologues are broadly distributed across eukaryotic
phyla. The yeast genome encodes homologues OSH1–7 that
are sorted into four subfamilies [19]. Entire or partial genomic
complements of OSBP homologues have been identified in
flies (Drosophila melanogaster) [20], plants [21,22], worms
(Caenorhabditis elegans) [23] and slime mould (Dictyostelium
discoideum) [24]. At one extreme, the parasitic protist
Cryptosporidium parvum expresses only two ORPs, a short-type
containing only the ligand-binding domain and a longer variant
with an N-terminal PH domain [25], whereas at the other end of
the spectrum Arabidopsis thaliana and mammals have 12 genes
[14]. Phylogenetic relationships between 120 taxa revealed
support for clustering on the basis of type rather than taxonomic
distribution, suggesting a set of early evolutionary ancestors
[14,25]. Groupings of 2–3 mammalian and fungal OSBP/ORPs
within each cluster also indicate more recent duplication events.
Interestingly, the presence of OSBP/ORPs in D. melanogaster
c The Authors Journal compilation c 2010 Biochemical Society
and C. elegans, neither of which synthesize sterols, implies a
lack of involvement in regulation of endogenous synthesis [26].
However, this does not preclude a role in uptake or transport as
these organisms require exogenous sterols for survival.
Owing to the size of the gene family and diversity of promoter
and splice variants, ORPs have highly complex expression
patterns that have not been correlated with tissue-specific
functions. Studies credited with the initial profiling of the human
and murine ORP gene family reported the tissue distribution
of mRNA transcripts by Northern blotting, hybridization with
human-tissue-expression filter arrays [12,13] and RT (reverse
transcription)–PCR analysis [13,14,27]. Although OSBP/ORP
mRNAs are expressed to some extent in most human tissues,
transcript levels vary significantly between tissues and within
subfamilies. For instance, OSBP (subfamily I) is expressed in a
variety of human and mouse tissues, but predominantly in brain,
heart, kidney and liver, whereas transcripts for ORP4L and ORP4S
(also subfamily I) are expressed almost exclusively in brain and
heart [13,15,27]. Similarly, protein and mRNA expression profiles
of subfamily III members are tissue-specific and non-overlapping:
ORP3 is expressed in kidney, lymph nodes and thymus; ORP6
is in brain and skeletal muscle; and ORP7 is restricted to the
gastrointestinal tract [28]. It is also notable that OSBP/ORP gene
expression profiles are not shared among tissues with a prominent
role in sterol metabolism and, with the exception of ORP6 [13] and
ORP1 [12], are not regulated by altering cellular sterol levels. The
lack of acute regulation of OSBP/ORP gene expression suggests
essential housekeeping function(s).
STRUCTURAL ORGANIZATION OF OSBP/ORPs
OH/sterol-binding domain
The defining feature of the OSBP/ORP gene family is the
aptly named C-terminal 400 amino acid OH domain that
binds cholesterol and oxysterols [29–32] (Figure 1). The
sterol-binding activity of OSBP/ORPs are routinely assayed by
dispersing oxysterols or cholesterol in aqueous buffers or with
low concentrations of detergent respectively [29,31]. Under
these conditions, OSBP binds 25-OH (25-hydroxycholesterol)
(K d of 10 nM) and cholesterol (K d of 173 nM) with relatively
high affinity [15,33–35]. Competition-based assays revealed
that OSBP binds other oxysterols with reduced affinity and
does not bind steroid hormones [33]. Similarly, ORP4L
binds 25-OH (K d of 10 nM) in vitro [15], an activity that
is inhibited by mutations in the putative lid region of the
binding fold [31]. Despite the binding of cholesterol (K d of
267 nM) and 25-OH (K d of 48 nM) by a GST–ORP4S fusion
protein, encompassing the FFAT and sterol-binding domain
[31], ORP4S expressed in CHO (Chinese-hamster ovary) cells
was devoid of sterol-binding activity perhaps due to a cellular
inhibitory factor [15]. ORP2 binds 25-OH (K d of 3.9 μM),
(22R)-OH [(22R)-hydroxycholesterol] (K d of 14 nM),
7-ketocholesterol (K d of 140 nM) and cholesterol [32,36].
ORP1L binds (22R)-OH and 25-OH [32,37] and the OH domain
of ORP8 bound 25-OH but not (24S)-hydroxycholesterol or
7-oxocholesterol [38].
The sterol-binding activity of OSBP/ORPs has also been
demonstrated in cultured cells by using photo-activatible 6-azicholesterol and -25-OH [32]. The OH domains of OSBP and
ORPs 1–5, 7 and 10 expressed in COS cells formed adducts
with photo-25-OH as did full-length ORPs. With the exception
of ORP9 and ORP4, all family members formed adducts with
photo-cholesterol. Although the binding constants and ligand
specificity are not available for the entire family, it is apparent that
Oxysterol-binding protein gene family
OSBP/ORPs bind cholesterol and oxysterols in the low nM range
and display preference for side-chain hydroxylated sterols, such
as 25-OH. As the apparent affinity for oxysterols and cholesterol
differs only 3–4-fold and cholesterol is more than 103 -fold more
abundant than oxysterols, the endogenous ligand for OSBP/ORP
could be cholesterol. However, the difference in solubility of these
sterols suggests that OSBP/ORPs could preferentially bind to
oxysterols in the cytoplasmic compartment and to cholesterol in
membranes.
The structure of the OH domain of the yeast Osh4p has been
solved in complex with cholesterol, ergosterol or oxysterols [39].
The general features of the binding fold, which accommodates
a single sterol ligand in a hydrophobic tunnel that is capped by
an α-helical lid, is consistent with a sterol transport function.
The ligand-binding core is formed by a 180 amino acid
β-barrel consisting of a 19-strand, anti-parallel β-sheet that forms
a hydrophobic tunnel with a hydrophilic exterior. The tunnel
is capped by a flexible N-terminal lid, followed by a twostranded β-sheet and three α-helices that form a wall of the
β-barrel, plugging the end of the tunnel distal to the lid. The
lid of Osh4p also contains a curvature-sensing activity similar
to the Arf (ADP-ribosylation factor)-GAP (GTPase-activating
protein) lipid packing sensor that might assist in selective
binding to Golgi membranes [40]. A 125 residue C-terminal
region localized to the distal end of the β-barrel is implicated
in membrane binding and sterol transfer [41]. Ergosterol,
cholesterol, 7-hydroxycholesterol, 20-hydroxycholesterol and
25-OH are positioned with the 3-hydroxy groups at the bottom
of the tunnel and the sterol iso-octyl group interacting with
side chains in the lid by van der Waals interactions. Quite
unexpectedly, sterol hydroxy groups do not make direct contact
with residues lining the tunnel, but are hydrogen-bonded to
polar residues via water molecules. Two water molecules are
bound to the sterol 3-hydroxy group and Gln96 , which is in a
cluster of hydrated residues (Trp46 , Tyr97 , Asn165 and Gln181 ), at
the bottom of the tunnel. The 25-hydroxy side chain of 25-OH
interacts with Lys109 at the mouth of the tunnel via two water
molecules, but other oxysterol hydroxy groups do not demonstrate
such ordered interactions. Interestingly, Osh4p could only be
crystallized either as a deletion mutant, lacking the N-terminal lid,
or in a ligand-bound form, indicating that the N-terminal region
is highly flexible and disordered in the apo-state [39]. Molecular
dynamics simulations of the binding and release of cholesterol
suggest that Osh4p undergoes significant conformational changes
between bound and apo forms, consistent with opening and
closing of the lid [42,43].
PH domains
Full-length ORPs account for approx. 4 % of all PH domaincontaining proteins encoded by the human genome [44]. Despite
limited sequence similarity, all PH domains consist of a conserved
seven stranded β-sandwich capped by an α-helix. Opposite to
the helical cap, conserved basic residues in loops connecting the
β-strands mediate interaction with compositionally diverse PIPs
found in eukaryotic membranes. However, less than 10 % of PH
domains bind PIPs with high affinity and specificity [45] and
only 30 % of PH domain-containing proteins in yeast display
membrane targeting [44].
PH domains are distinguished from other PIP-binding
domains by their specific high-affinity binding to PIPs with
two vicinal phosphate groups: PtdIns(3,4)P2 , PtdIns(4,5)P2 or
PtdIns(3,4,5)P3 [45]. This specificity results in targeting of some
eukaryotic PH domain proteins to the PM (plasma membrane)
where PIP levels are increased as a result of signalling events.
15
Among a handful of outliers in this group are the closely related
OSBP, Osh1p and Osh2p PH domains that bind PtdIns4P and
PtdIns(4,5)P2 with similar, moderate affinity in vitro. In vivo,
however, OSBP and Osh1p PH domains specifically localize to the
Golgi apparatus in a PtdIns4P-dependent manner, a feature that
has led to their use as diagnostic tools for qualitative measurement
of PtdIns4P in the Golgi apparatus and other membranes [46,47].
Golgi localization of the OSBP and Osh1p PH domains in
yeast is dependent on Arf1p and PtdIns4P synthesis by Pik1p
(phosphatidylinositol kinase 1) [47]. It was later confirmed
that mammalian ARF1 is required for membrane targeting of
the OSBP PH domain [48]. Conserved basic residues in the
variable β-loops of the OSBP PH domain are required for PIP
binding; however, a second site for Golgi interaction in the
Osh1p PH domain involves a histidine residue at position 79 in
the β7 strand [49]. This surface of the PH domain is positioned
away from the plane of the membrane and thus probably interacts
with a protein factor, possibly Arf1. The presence of an arginine
residue at this position in the Osh2p PH domain results in affinity
for PtdIns4P at the Golgi [49] and PM [50]. Interestingly, His79 is
conserved in OSBP and ORP9, both of which bind PtdIns4P
and are localized to the Golgi apparatus [29,51], suggesting
a conserved two-site interaction model for Golgi-specific PH
domains that involves lipid and protein receptors.
The PH domains of other mammalian ORPs also bind PIPs
but are poorly characterized with respect to ligand specificity
and in vivo membrane targeting. The PH domain of ORP3,
which binds PtdIns(3,4)P2 and PtdIns(3,4,5)P3 with moderate
affinity [52], is necessary, but not sufficient, for PM localization
[28,52]. The ORP1L PH domain binds liposomes containing
PtdIns(3,4)P2 , PtdIns(3,5)P2 and PtdIns(3,4,5)P3 with low affinity
and specificity, however PH domain-dependent localization is not
observed [53].
Interestingly, several short ORPs lacking PH domains display
in vitro PIP-binding activity. Residues 205–314 of Osh4p,
corresponding to β-strands 9–17 of the sterol-binding fold, bound
PtdIns(4,5)P2 in vitro with affinity similar to the full-length Osh4p
[54]. Mutations in this region that inhibit PIP-binding also prevent
Golgi localization of Osh4p in yeast cells [54]. A similar region of
ORP1S binds a variety of PIPs, but, unlike full-length ORP1S and
Osh4p, this region was not sufficient to inhibit Sec14 (secretory
14)-dependent Golgi secretion in yeast [55]. Other short ORPs,
including Osh6p, ORP2, ORP10S and ORP9S, non-specifically
bind PIPs and other anionic phospholipids that are immobilized
on membrane supports [56–58]. In addition, Osh4p also catalyses
the extraction and transfer of PtdIns(4,5)P2 from liposomes [59].
This activity is less efficient than sterol extraction and transfer, but
is unaffected by mutations that block these activities, suggesting
that a second site is involved that is distant from the sterolbinding region. The aforementioned PtdIns(4,5)P2 -binding region
of Osh4p comprises two positively charged loops opposite the
sterol-binding pocket that mediate second-site binding of anionic
lipids required for sterol transfer by a mechanism involving
simultaneous contact between adjacent membranes [41].
FFAT motif and VAP binding
Mammalian ORPs of subfamilies I, II, III and IV contain a
FFAT motif (E-F/Y-F/Y-DAxE) that binds mammalian VAP-A
and VAP-B or, in the case of yeast Osh proteins, Scs2p (suppressor
of choline sensitivity 2) [15,17,60,61] (Figure 1). VAP is a
type II integral membrane protein that is anchored to the
ER by a C-terminal hydrophobic domain that resembles that
of tail-anchored proteins that are inserted post-translationally
[62]. The cytoplasmic domain consists of an N-terminal MSP
c The Authors Journal compilation c 2010 Biochemical Society
16
M. H. Ngo, T. R. Colbourne and N. D. Ridgway
(major sperm protein)-like motif that binds FFAT and a
coiled-coiled domain involved in multimerization. The combination of FFAT and PH domains provides most OSBP/ORPs with
dual membrane-targeting activity. However, this relationship does
not strictly apply: ORP2 and ORP9S lack PH domains but have
FFAT motifs; ORP1S and ORP4S lack both; and ORP10 and
ORP11 have only PH domains. In lieu of a FFAT motif, subfamily
IV ORPs are anchored to the ER by C-terminal transmembrane
domains [38]. Interestingly, 14 out of 17 eukaryotic proteins
containing perfect FFAT motifs have been implicated in lipid
metabolism, indicating that VAP is an important regulator of lipid
homoeostasis in the ER and other organelles [61,63]. For a more
in-depth analysis of other biological functions of VAP see [64].
Ankyrin domain targeting to membrane contacts
ORP1, Osh1p and Osh2p each contain three ankyrin motifs
(Figure 1), a common 33-residue structural domain comprised of
two antiparallel α-helices separated by a β-hairpin, which forms
a protein–protein interaction surface (reviewed in [65]). Osh1p
is uniquely localized to the NVJ (nuclear–vacuolar junction),
a site of piecemeal autophagy of the nucleus [66], and the
TGN (trans-Golgi network) [50]. The sequestration of Osh1p
to the NVJ requires interaction of the three ankyrin repeats
with Nvp1 (nuclear vacuolar protein 1), which anchors the
outer nuclear membrane to the vacuole through interactions
with Vac8 (vacuole-related 8) [50,67]. Although Osh1p is not
required for NVJ formation, its partitioning between the Golgi
and NVJ appears to be important for raft-dependent trafficking of
the tryptophan transporter, Tat2p [68]. Osh2p also has a threeankyrin-repeat motif, but does not localize to the NVJ, nor
does an Osh1p chimaera containing the Osh2p ankyrin repeats
[50]. The only mammalian ORP with three ankyrin repeats,
ORP1L, does not localize to NVJs but could form cholesteroldependent ER–endosome contact sites [69]. The ankyrin motifs
in ORP1L interact with Rab7 to regulate endosomal positioning
on microtubule tracks [53,70].
BIOLOGICAL FUNCTIONS OF OSBP/ORPS
OSH cells implied an important role in sterol regulation, but it
was not until the structure of Osh4p was solved that it became
apparent that the family might have a conserved transfer function
[39]. Thereafter, Raychaudhuri et al. [59] showed that Osh4p
catalysed the non-vesicular transport of cholesterol and ergosterol
between liposomes in vitro, an activity that was enhanced 2- to
4-fold by inclusion of PtdIns(4,5)P2 in donor and/or acceptor
liposomes. All OH domains of the OSH family are capable of
transferring cholesterol in vitro, but Osh2p, Osh4p and Osh5p
are the most active when phosphatidylserine was included in
the donor and acceptor liposomes [41]. Osh4p displayed in vivo
transfer activity in an esterification-based assay, which monitored
cholesterol and ergosterol transfer from the PM to the ER,
but it was evident that other Osh proteins were also involved,
particularity Osh3p and Osh5p [59]. Even though Osh4p is not
localized to the ER or PM [54], its expression can partially restore
PM-to-ER sterol transport. This suggests that individual Osh
proteins are loosely sequestered at one or more organelles, but
can function outside these constraints to compensate for the loss
of other family members.
ER-to-PM cholesterol transport in yeast has a nonvesicular component that involves equilibration of non-raftassociated sterols between the two compartments [71]. Although
sphingolipids control this bidirectional pathway by influencing the
free sterol pool in the PM, an OSH strain had a 5-fold reduction
in ergosterol transport to the PM [72,73]. It was speculated that a
sterol transfer function was not consistent with the reported roles
of Osh proteins in vesicular trafficking associated with polarized
cell growth. Inactivation of OSH genes results in lack of Rho1p
and Cdc42p localization and defective vesicle fusion at bud sites,
a defect that is rescued by expression of any OSH gene [74].
However, it is conceivable that Osh proteins regulate the localized
sterol environment at bud sites, and other regions of the PM, by
direct or indirect sterol transfer and thus influence the recruitment
and activity of lipid-anchored GTPases. The prevailing evidence
that Osh proteins bind and transfer sterols in vitro, collectively
transport sterols between the PM and ER, and that disruption of
Osh4p sterol- or phospholipid-binding activity abrogates rescue
of the OSH strain, supports the concept that the yeast family are
sterol transfer proteins [10,41,59].
Sterol transport by OSBP/ORPs
Mammalian OSBP/ORPs have a common cholesterol and/or
oxysterol-binding activity, yet individual members of the family
have seemingly disparate functions, such as regulation of
lipid metabolism, signal transduction, vesicular transport and
cytoskeletal regulation. These activities could be secondary to a
common sterol transport activity or primary signalling functions
that result from conformational changes induced by cholesterol or
oxysterol binding. Owing to the size of the OSBP/ORP family and
the potential diversity of sterol ligands, these proposed functions
need not be mutually exclusive. However, recent evidence
demonstrating in vitro and in vivo sterol transfer by OSBP/ORPs
and Osh proteins, and supporting structural evidence, suggests
that sterol transfer is a primary function which effects a variety of
downstream targets by altering membrane environment
Sterol transfer in yeast
The absence of other putative transfer proteins in yeast, such as
those of the START family, suggests that OSH proteins have key
sterol regulatory or transfer functions. Indeed, deletion of all seven
OSH genes (OSH) is lethal but bypassed by the expression of
any one OSH gene indicating common/related function(s) [19].
The substantive accumulation of ergosterol and other sterols in
c The Authors Journal compilation c 2010 Biochemical Society
Sterol transfer by mammalian OSBP/ORPs
Recent evidence has also suggested that OSBP and several ORPs
have sterol transfer activity that could underlie the observed
effects of these proteins on lipid and sterol homoeostasis. OSBP
was originally hypothesized to inhibit cholesterol synthesis by
interacting with 25-OH. However, stable expression in CHO
cells did not affect 25-OH-mediated suppression of HMG-CoA
synthase, HMG-CoA reductase or LDLR [LDL (low-density
lipoprotein) receptor] mRNAs [29]. Moreover, silencing of OSBP
expression by RNAi (RNA interference) failed to affect sterolregulatory events at the ER [75,76].
OSBP localizes to the ER and Golgi apparatus via FFAT
domain interaction with VAP [17] and PtdIns4P binding by
the PH domain [30,46] respectively. Within these compartments,
OSBP stimulates SM (sphingomyelin) biosynthesis in response
to cellular cholesterol and oxysterol levels. Overexpression and
silencing studies show that OSBP is required for 25-OHand cholesterol-dependent stimulation of SM synthesis [75,77].
Activation occurs via recruitment of the CERT (ceramide transfer
protein) to the Golgi apparatus, resulting in increased ceramide
delivery to SM synthase [75]. Defective SM synthesis in OSBPdepleted cells was not rescued by expression of OSBP with
mutations in the PH, FFAT or sterol-binding domains, indicating
Oxysterol-binding protein gene family
Figure 2 Functional consequences of OSBP- and ORP9L-dependent sterol
transfer between the ER and Golgi apparatus
Directional transfer of cholesterol and/or oxysterol by OSBP and ORP9L maintains sterol
homoeostasis in the ER to trans -Golgi/TGN as well as downstream organelles. In the case of
OSBP, sterol transfer would optimize the membrane environment of a PI4K (PtdIns 4-kinase)
resulting in increased PtdIns4P synthesis, recruitment of CERT and increased ceramide delivery
for SM synthesis. The co-ordinated transfer and synthesis of sterols and SM in elements of
the late Golgi apparatus and endosomes would then influence downstream processes such as
lipid-raft assembly, sterol efflux and APP processing. In the case of ORP9L, cholesterol delivery
to the Golgi apparatus affects secretion and sterol levels in post-Golgi compartments.
a mechanism involving contacts between the ER and Golgi [75].
This initially suggested that OSBP binds cholesterol or oxysterol
in the Golgi and/or ER and uses this signal to increase SM
synthesis as a means of co-ordinating the level of these two
raft-associated lipids. This relationship is further strengthened
by the observation that depletion of SM or cholesterol at the PM
promotes dephosphorylation and translocation of OSBP to the
Golgi apparatus [78].
The lack of interaction between OSBP and CERT indicates
an indirect mechanism of activation, suggestive of steroldependent signalling. However, OSBP could also function as a
sterol transfer protein between the ER and Golgi that modifies
the membrane environment and Golgi recruitment of CERT
(Figure 2). Consistent with this possibility, we recently showed
that OSBP not only binds sterols presented in aqueous and detergent dispersion, but also extracts cholesterol from liposomes and
mediates its transfer between donor and acceptor liposomes [51].
Cholesterol transfer is stimulated by PtdIns4P binding by the PH
domain, reproducing at least one stage of cholesterol transfer
between the Golgi apparatus that involves membrane-specific
targeting [51]. Unlike Osh4p, PtdIns4P stimulated cholesterol
transfer by OSBP when included only in donor liposomes, both
donor and acceptor liposomes, but not in acceptor liposomes
alone. This could reflect a requirement for both cholesterol
and PtdIns4P in the same membrane and shows that PtdIns4P
imposes some degree of directionality on cholesterol transfer.
Demonstration of sterol transfer activity by OSBP provides a
mechanistic basis to explain CERT activation as follows: OSBP
interaction with VAP in the ER would facilitate binding of
cholesterol (or oxysterols), followed by targeting to the Golgi
apparatus and release of sterol ligand; the subsequent enrichment
of sterols in the Golgi apparatus would alter the membrane
environment leading to CERT recruitment (Figure 2). Current
17
evidence points towards a sterol- and OSBP-dependent activation
of PtdIns4P synthesis in the Golgi, leading to increased CERT
recruitment via its PH domain [79,80].
OSBP in the ER/Golgi also appears to directly or indirectly
affect sterol-dependent activities in other areas of the secretory
pathway. Cholesterol efflux at the PM by the ABC (ATP-binding
cassette) A1 transporter protein is increased 3-fold in CHO
cells depleted of OSBP [81]. Down-regulation of ABCA1 by
OSBP overexpression was prevented by disruption of cholesterol
binding, but not by mutations that abolished localization to the
Golgi apparatus or ER [81]. As OSBP regulation of CERT activity
requires the PH, FFAT and sterol-binding domains, inhibition
of ABCA1 expression appears to be independent of its role in
sterol-activated SM synthesis in the Golgi apparatus. Similarly,
OSBP expression is negatively correlated with processing of
APP (amyloid precursor protein) and secretion of amyloidogenic
Aβ (amyloid β-peptide) [82]. OSBP overexpression caused
accumulation of APP–Notched dimers in the Golgi apparatus,
an effect that was reversed by addition of 25-OH. As Aβ
levels are positively correlated with cholesterol [83,84], OSBP
could reduce the sterol composition of membranes where APP
processing occurs and attenuate Aβ production. Although it
remains to be determined where and how OSBP exerts these
effects, these results implicate OSBP in the control of cholesterol
or oxysterol distribution in Golgi and post-Golgi compartments,
thus regulating the activity of cholesterol-sensitive functions, such
as secretases and ABCA1 (Figure 2).
Similar to OSBP, ORP9L localizes to the trans-Golgi/TGN
and ER via its PH domain and FFAT motif respectively,
and catalyses PtdIns4P-dependent cholesterol transfer between
liposomes [17,51]. The sterol-binding and transfer activity of
ORP9L was not linked to SM metabolism, and Golgi localization
of ORP9L was not affected by oxysterols or altered cholesterol
homoeostasis [17,51]. Silencing of ORP9L expression in CHO
cells resulted in accumulation of cholesterol in filipin-positive
endosomes/lysosomes but did not affect cellular cholesterol mass
or synthesis, suggesting that cholesterol movement between the
ER and Golgi apparatus is ultimately affecting other downstream
compartments. Interestingly, ORP9L depletion also fragmented
the Golgi apparatus. The TGN is an important delivery site
for endocytosed cholesterol [85] and cholesterol overload or
depletion strongly inhibits secretory activity [86–88]. Thus
transport of cholesterol between the trans-Golgi/TGN and ER by
ORP9L could be required to maintain optimal cholesterol levels
in the secretory pathway (Figure 2).
Initial overexpression experiments showed that ORP2 increased
cholesterol efflux to acceptors in the medium and decreased cholesterol esterification and increased synthesis, possibly due
to Golgi localization [57,90]. However, ORP2 was recently
shown to undergo sterol-dependent movement between the
cytoplasm and lipid droplets; an ORP2 sterol-binding mutant
was constitutively associated with lipid droplets but 22hydroxycholesterol inhibited the association [36]. Silencing of
ORP2 expression by RNAi decreased TAG (triacylglycerol)
hydrolysis and increased cholesterol ester formation, but the
latter effect was only observed when lipid droplet formation
was enhanced by oleate treatment [36]. The sterol-dependent
localization of ORP2, coupled with VAP- and PIP-binding
activity, is suggestive of a transfer activity that controls neutral
lipid synthesis in lipid droplets.
Mechanisms of sterol transport by the OSBP gene family
As OSBP/ORPs have the potential to interact with donor and
acceptor membranes by independent targeting functions, an
c The Authors Journal compilation c 2010 Biochemical Society
18
M. H. Ngo, T. R. Colbourne and N. D. Ridgway
Figure 3 Mechanisms of sterol transfer and integration with sterol sensing
functions
(A) Sterol transfer by OSBP/ORPs could involve sequential binding to donor and acceptor
membranes. In this model, OSBP/ORPs interact with VAP (dark blue) at the ER to acquire sterol,
followed by dissociation and release of sterol ligand after attaching to an acceptor membrane via
a PH domain-dependent interaction with PIPs (green). Intramolecular interactions between the
OH/sterol-binding and PH domains, or reversible phosphorylation, could promote directional
transfer. (B) Sterol transfer could also proceed by tethering at membrane contact sites through
simultaneous PH and FFAT domain interactions or, in the case of ORPs lacking PH and/or
FFAT domains, via dual interactions with anionic lipids (PIPs and phosphatidylserine). (C) In
a variation of the contact site model, the PH domain of ER-tethered ORP8 could interact with
target membranes in cis or trans to facilitate sterol transfer or signalling. In all three scenarios,
associated signalling functions could be mediated by interaction of partner proteins with apo
and/or holo OSBP/ORPs (indicated in boxes, see the text for details). An animated version of
this Figure is available at http://www.BiochemJ.org/bj/429/0013/bj4290013add.htm.
important question is how the sterol-loaded protein transverses the
gap between membranes [91,92]. This could involve: (i) complete
disengagement of OSBP/ORPs from membranes after binding
and releasing sterol ligands (Figure 3A) or (ii) transfer while
OSBP/ORPs are simultaneously engaged with both donor and
acceptor membranes at contact sites (Figures 3B and 3C). The
membrane contact site model is particularly alluring as most
OSBP/ORPs have FFAT and PH domains that could mediate
c The Authors Journal compilation c 2010 Biochemical Society
simultaneous contact with two closely apposed membranes. Sterol
transfer is then predicted to occur by flip-flop of the sterolbinding domain between donor and acceptor membranes.
Close apposition of the ER and trans-Golgi cisternae, where
OSBP and ORP9L are predicted to localize, as well as other
organellar membranes, has been identified by electon microscopy
ultrastructure studies [93,94]. With the exception of ORP1L (see
below), OSBP/ORPs have not been localized to, or directly
implicated in, the formation of membrane contact sites, nor is
there evidence that sterol transport occurs at contact sites. In
the absence of PH and FFAT domains, OH domains from all
Osh proteins form simultaneous contacts between two liposomal
membranes enriched in anionic lipids that, in the case of Osh4p,
greatly enhanced sterol transfer [41]. Mechanistically this could
involve dual membrane contacts at the entrance of the sterolbinding pocket and a distal lipid-binding site that would facilitate
sterol uptake, followed by disengagement or ‘pivoting’ to deliver
ligand to the acceptor membrane (Figure 3B). Even if membranes
are sufficiently separated to prevent simultaneous contact, yeast
or mammalian OSBP/ORPs could be concentrated at these sites
where collisional transfer would be favoured. Figure 3(C) shows
a potential variation of the contact site model wherein the transmembrane domain of ORP5 or 8 anchor the proteins to the
donor site and the PH domain interacts with PIPs on the target
membrane.
Another important consideration is the directionality of sterol
transfer between donor and acceptor membranes. As an example,
vectoral transfer of ceramide by CERT between two membranes is
regulated by phosphorylation-dependent conformational changes.
CERT contains a serine-rich phosphorylation site, adjacent
to the PH domain, which negatively regulates its interaction
with Golgi PtdIns4P [80]. Initial phosphorylation of CERT by
PKD (protein kinase D) in the Golgi apparatus is followed
by phosphorylation at nine adjacent serine residues by CK1
(casein kinase 1) γ 2, resulting in reduced Golgi localization,
ceramide transport and SM synthesis [95,96]. In the ER, protein
phosphatase PP2Cε (protein phosphatase 2Cε) interacts with
VAP and dephosphorylates CERT leading to increased Golgi
localization and SM synthesis [97]. Collectively, this explains
the ATP-dependence of ceramide transport [79] and shows that
an ER and Golgi phosphorylation cycle drives the delivery of
ceramide from the ER to the Golgi apparatus [98].
Given its close metabolic relationship to CERT it is feasible
that OSBP is under a similar control by phosphorylation. Indeed,
OSBP is also Golgi-localized and dephosphorylated in response
to depletion of cellular SM and cholesterol [78,99]. The relevant
OSBP phosphorylation site has multiple CK1 sites but is adjacent
to the FFAT motif and does not contain a PKD consensus
phosphorylation site. Independent of phosphorylation, OSBP
binding of exogenous oxysterol in cultured cells results in Golgi
localization, suggesting a conformational change that exposes the
PH domain and increases interaction with Golgi PtdIns4P and
other determinants [29,99] (Figure 3A). This could be a general
mechanism as an ORP3 N-terminal/OSBP C-terminal chimaera
showed increased localization to the PM in cells treated with
25-OH [52]. It remains to be determined how phosphorylation
and sterol-induced conformational changes are related to steroltransport between membranes.
Sterol-dependent signalling by OSBP/ORPs
It is difficult to strictly segregate putative sterol-transport
functions of OSBP/ORPs from those that are signallingrelated. One could envision a scenario whereby sterol binding
by OSBP/ORPs during transport to a target membrane also
Oxysterol-binding protein gene family
induces conformational signals that promote activation of
ancillary signalling pathways (Figure 3). This would conveniently
integrate cholesterol and oxysterol ‘sensing’ by growth regulatory
pathways with intracellular cholesterol transport and distribution.
Whereas conformational changes are induced by sterol binding
to Osh4p [39], these are more consistent with the opening and
closing of the binding pocket during ligand binding, a conclusion
supported by molecular dynamic modelling [42,43]. Although
there are differences in the lid region of Osh4p bound to side-chain
oxysterols compared with cholesterol, sterol ligands of Osh4p
are buffered from direct interaction with amino acid residues
by water [39]. This argues against a signalling mechanism
that differentiates between apo and holo forms of the sterolbinding domain, but the possibility that sterol binding triggers
subtle conformation effects that promote independent signalling
cascades cannot be excluded.
An example of this apparent dual function is OSBP, which
was identified as a sterol-sensing scaffold that regulates the
dephosphorylation and inactivation of ERK (extracellular-signalregulated kinase), a component of the MAPK (mitogen-activated
protein kinase) pathways [34,100]. The cholesterol-bound form
of OSBP is a scaffold for the serine/threonine phosphatase PP2A
and HePTP (haematopoietic tyrosine phosphatase), both of which
dephosphorylate and inactivate ERK. Removal of cholesterol or
binding of 25-OH to OSBP causes the phosphatase complex
to dissociate, allowing hyperphosphorylation and activation of
ERK [100]. Deletion mapping and pulldown experiments showed
that PP2A and HePTP bind at non-overlapping sites in the Cterminal 400 amino acids of OSBP [34]. A model was proposed
wherein cholesterol extraction from membranes by a CRAC
(cholesterol recognition/interaction amino acid consensus) motif
[101], which encompasses the α-helical lid and β-sheets 1–3
of the predicted binding fold [39], in conjunction with the Nterminal glycine/alanine-rich region and PH domain, induced
a conformation favourable for phosphatase recruitment. This
process could be envisioned as one step in a sterol transfer pathway
(Figure 3A).
Most of the experiments linking OSBP to ERK regulation
were performed in vitro or by overexpression in cultured cells.
However, phosphorylation of ERK was also decreased in liver and
cultured hepatocytes infected with an adenovirus encoding OSBP,
and was increased by silencing OSBP expression in hepatocytes
[102]. Adenoviral OSBP overexpression in murine liver elevated
plasma VLDL (very-low-density lipoprotein), TAG and mRNA
for Insig-1 and SREBP-1c, whereas RNAi suppressed insulinmediated SREBP-1c processing and decreased TAG synthesis.
This suggests that OSBP could regulate insulin-dependent TAG
metabolism by modulating ERK activity.
In an apparently unrelated role that involves a similar
scaffolding mechanism, stimulation of pro-atherogenic profilin-1
expression in endothelial cells by 7-oxocholesterol involves
JAK2 (Janus kinase 2) phosphorylation of Tyr394 in OSBP
and subsequent recruitment and phosphorylation of STAT3
(signal transducer and activator of transcription 3) [103]. This
provides a mechanism for oxysterol-mediated dysfunction of
the cytoskeleton in endothelial cells but seems at odds with the
relatively low affinity of OSBP for side-chain hydroxylated sterols
[104,105].
In addition to a role in regulation of ER–Golgi cholesterol
transport, ORP9L has been implicated in the regulation of
Akt activity, raising the possibility that sterol transfer could
be integrated with this important proliferative pathway. ORP9L
and ORP9S contain a PDK2 (phosphoinositide-dependent kinase
2) phosphorylation site, termed the hydrophobic motif, that is
required for activation of AGC-type kinases (protein kinase
19
A/protein kinase G/protein kinase C-family kinases), such as Akt
[106]. In bone marrow mast cells, the hydrophobic motifs of
ORP9S and Akt were co-ordinately phosphorylated, and RNAidepletion of ORP9L in HEK (human embryonic kidney)-293 cells
resulted in a 3-fold increase in phosphorylation of the Akt PDK2
site, but not the PDK1 site. [106]. These results suggest that
ORP9 is a negative regulator of Akt phosphorylation, but whether
this is a direct interaction or secondary to altered sterol distribution
is unknown.
Both ORP1L and ORP8 affect expression of ABCA1 and other
LXR target genes by a possible signalling-related mechanism.
Gene silencing of ORP8, by RNAi, increased ABCA1 expression
in macrophages via up-regulation of LXR promoter activity [38].
As ORP8 is an ER transmembrane protein, this could occur by
transport at membrane contact sites, via an intermediary sterol
carrier(s) or by altered synthesis of LXR ligands in the ER
(Figure 3C). Interestingly, ABCA1 and ORP8 are reported to
co-immunoprecipitate suggesting that interaction in the ER could
be negatively regulating ABCA1 expression and activity [89].
Overexpression of ORP1L enhanced [53,70] or repressed [37] the
activity of LXR and expression of some target genes, indicating
a cell-specific effect at the levels of sterol-transfer or signalling
functions.
Evidence for involvement of OSBP/ORPs in cell proliferation,
survival and differentiation has also been derived from other
single and multicellular animal and plant models. A D.
melanogaster OSBP homologue (OSBP-Dm) prevented Wee1p
inhibition of p34cdc2 and cell-cycle progression when expressed
in Schizosaccharomyces pombe [20]. As D. melanogaster does
not synthesize sterols, it was speculated that OSBP-Dm has a
cell cycle/cell signalling function. In C. elegans, a truncated ORP
missing the PH domain was identified as a novel regulator of the
TGF-β (transforming growth factor-β) signalling pathway based
on interaction with human BRAM [BMP (bone morphogenic
protein) receptor-associated protein] [23]. Inhibition of BIP
(BRAM-interacting protein) expression by RNAi phenocopied
TGF-β deletion and affected body length, suggesting that this
ORP might have a broader role in C. elegans TGF-β signalling
pathways. During D. discoideum culmination (the transition from
the slug to a fruiting body) the expression of Dd-OSBPa was
increased and enriched in the periphery of pstO cells (the rear
two-thirds of a slug) [24]. The formation of slugs was normal in
OSBPa-null cells, however the culmination–fruiting body switch
was defective. A screen linked to signalling functions lead to
the identification of PiORP1 (Petunia inflata oxysterol bindingprotein-related protein 1) as an interacting partner and substrate
for receptor-like kinase PRK1 (pollen-expressed receptor-like
kinase), an essential kinase in pollen development [22]. PiORP1
and PRK1 expression coincide at the pollen development stage,
but persist into later stages of plant growth. A salt-inducible OSBP
homologue from soyabean (Glycine max) termed GmOSBP
is expressed highly during middle–late phases of cotyledon
senescence and could be involved in growth and the stress
response [107]. Similarly, the expression of a potato OSBP
homologue lacking a PH domain is up-regulated following
exposure to Phytophthora infestans, the cause of late blight [21].
These studies point to important roles for OSBP/ORPs in diverse
organisms but require further investigation to identify ligands and
differentiate signalling from lipid transport/regulatory functions.
OSBP/ORP interaction with the cytoskeleton
An emerging theme in this field is the interaction of OSBP/ORPs,
as well as their cognate binding partner VAP, with the three
classes of cytoskeletal elements: actin, intermediate filaments
c The Authors Journal compilation c 2010 Biochemical Society
20
M. H. Ngo, T. R. Colbourne and N. D. Ridgway
and microtubules. Ultrastructural analysis of mouse hippocampal
slices revealed that VAP was associated with the ER and
microtubules and was frequently positioned between these two
structures [108]. Interaction of VAP with microtubules was
required for regulation of ER membrane protein lateral diffusion
[109] and bouton formation at neuromuscular junctions in
D. melanogaster [110]. The regulation of microtubule
organization at neuromuscular junctions is particularly interesting
because of the recent identification of a rare form of motor neuron
disease/ALS8 (amyotrophic lateral sclerosis 8) that is caused by
a VAP-B P56S mutation [111–113]. The physical interaction of
VAP with microtubules appears to involve the FFAT domain, but
could occur directly or indirectly through other FFAT-containing
proteins, such as Nir2 [109,114].
A series of studies show that ORP1L is a link between
VAP and the microtubule network. ORP1L recruits microtubule
motors on to LEs (late endosomes)/lysosomes through ankyrin
interactions with the GTP-bound form of Rab7 to promote
clustering of LE/lysosomal compartments [53,70]. On the basis
of FRET (fluorescence resonance energy transfer) and pulldown
experiments, Rab7 recruits RILP (Rab7-interacting lysosomal
protein) and ORP1L to LE/lysosomes to form a tripartite complex
[70]. The complex then recruits p150Glued , a component of the
dynein–dynactin motor complex, which in turn drives minusend microtubule transport of the LE/lysosomes. The cholesterol
content in the outer membranes of LEs regulates the recruitment
of the dynein–dynactin–p150glued complex by a sensing function of the ORP1L sterol-binding domain [69]. When endosomal
cholesterol is elevated, ORP1L binds cholesterol and promotes
minus-end transport and clustering of endosomes around the
microtubule-organizing centre, a situation that is observed in
NPC (Niemann–Pick C) lysosomal cholesterol storage disease.
Depletion of endosomal cholesterol enhances the interaction
of ORP1L with its ER partner VAP, subsequently displacing
dynein–dynactin–p150glue and dispersing LEs. The interaction
between ORP1L and VAP seems to occur at ER–LE contact sites
(Figure 3B), but an increase in contact sites under cholesteroldepleted conditions and the lack of effect of ORP1L on LE
cholesterol levels lead the authors to conclude that ORP1L is
primarily a cholesterol sensor [69].
Overexpressed ORP10 co-localizes with microtubules
and induces a cable-like morphology that was dispersed by
depolymerizing agents [115]. Silencing of ORP10 in hepatoma
cells increased cholesterol and TAG synthesis and VLDL
secretion, cellular functions that could be related to its genetic
linkage to high TAG traits in a Finnish cohort [115]. This prompted
speculation that the ORP10 interaction with microtubules could
be linked to organelle positioning and secretion.
The intermediate filament protein vimentin is implicated in
esterification and transport of LDL-derived cholesterol [116,117].
When overexpressed in CHO cells, ORP4S caused the vimentin
network to collapse into thick bundles and inhibited esterification
of LDL-cholesterol, suggesting that vimentin–ORP4 interactions
could be involved in cholesterol delivery to the ER [15]. The
vimentin-binding site is within the sterol-binding domain of
ORP4, but a leucine repeat N-terminal of the FFAT motif, which
is absent in ORP4S, maintains the integrity of the intermediate
filament network [31]. OSBP with mutations in the leucine repeat
also collapsed the vimentin network, but this was via dimerization
with ORP4L and hence indirect. These novel interactions suggest
that the lipid regulatory and/or transportation functions of certain
ORPs can be mediated through interactions with the intermediate
filaments.
ORP3 affects the organization of the actin cytoskeletal network
through interactions with R-Ras [118]. ORP3 and R-Ras co
c The Authors Journal compilation c 2010 Biochemical Society
immunoprecipitated and are concentrated at filopodial protrusions
of HEK-293 cells. ORP3 overexpression inhibited integrin
activity and enhanced polarized cell protrusions, which was
dependent on PM-targeting by the PH domain. Other actinregulated functions were impaired by ORP3 overexpression in
primary macrophages, including cell spreading and podosome
formation. ORP3 silencing increased integrin activity, resulting
in enhanced spreading and extracellular matrix adhesion,
a phenotype accompanied by reorganization of the actin
cytoskeleton. Thus ORP3 seems to regulate cell adhesion,
spreading and migration by inhibiting the Ras signalling pathway,
which controls integrin activity and the actin cytoskeleton.
Interactions of ORPs with the cytoskeleton seem to fall into two
classes: direct physical interactions that affect the network organization (ORP4 and ORP10) and those that are secondary
to signalling-related mechanisms (ORP3 and ORP1L). It is
conceivable that the cytoskeleton is a scaffold on which OSBP/
ORPs transfer sterols between membranes that are contacted
by these networks. Alternatively, sterol-binding/transfer activity
could be integrated with these interactions as a means to impart
sterol-dependent regulation of the cytoskeleton (Figure 3). More
studies are required to determine whether this is a general feature
of the OSBP/ORP family, restricted to specific subfamilies or
members, or mediated by interaction with VAP.
INVOLVEMENT OF OSBP/ORPs IN HUMAN DISEASE
As OSBP/ORPs are implicated in regulation and transport of
cholesterol and oxysterols, which mislocalize or accumulate in
neurological and cardiovascular diseases, altered ORP expression
or activity might be expected to accompany disease onset and/or
progression. To date, human diseases that result from mutations
in OSBP/ORP genes have not been identified. This is not
surprising as functional redundancy within the family could
prevent the expression of phenotypes associated with mutations in
a single OSBP/ORP gene. However, evidence that OSBP/ORPs
are involved in dyslipidaemias continues to grow, indicating a
primary or secondary role in cellular sterol and lipid deposition.
OSBP/ORPs are frequently linked to LXR regulation of
cholesterol efflux and disposal, a pathway that affects formation
of arterial lesions resulting from accumulation of lipidladen macrophage-derived foam cells. Differentiation of human
monocytes into macrophages results in a >100-fold increase
in the ORP1L mRNA and appearance of the protein in the
endosomes/lysosomes. [16]. OSBP and other ORPs are not
similarly activated. Cholesterol-loading of human macrophages
with acetyl-LDL further increased ORP1L expression by 75 %
[37] and increased ORP6 mRNA by 2-fold [13]. The influence of
ORP1L expression on macrophage cholesterol metabolism was
examined by bone marrow transplant from macrophage-specific
ORP1L transgenics into LDLR−/− mice. Following the feeding of
a western-type diet, LDLR−/− mice expressing macrophages with
a 15-fold increase in ORP1L expression had a 2.1-fold increase
in aortic root lesion size despite having plasma lipid levels that
were similar to controls [37]. This was accompanied by reduced
expression of ABCG1, ABCG5 and ApoE (apolipoprotein E),
increased PLTP (phospholipid transfer protein) and interleukin1β, and reduced efflux of macrophage cholesterol to HDL (highdensity lipoprotein). ORP1L silencing in RAW224.7 cells also
inhibited the up-regulation of ABCG1 expression by (22R)OH treatment. Interestingly, overexpression of ORP1L in COS
or HEK-293 cells increased LXRα reporter gene activity in
response to (22R)-OH and non-sterol agonists, indicating that
cell- and oxysterol-specific responses are modulated by ORP1L
[16]. As ORP1L is a sterol-dependent regulator of endosomal
Oxysterol-binding protein gene family
positioning [69], it could be involved in cholesterol egress from
this compartment for conversion into the oxysterol activators of
LXR.
ORP8 is also involved in atherosclerotic lesion formation as
immunohistochemical analysis showed prominent staining of
ORP8 in lesional CD68+ macrophages compared with those
from a healthy arterial wall [38]. RNAi silencing of ORP8
in macrophages increased ABCA1 expression and cholesterol
efflux to ApoA1 (apolipoprotein A-I), was synergistic with a
synthetic LXR agonist and inhibited by mutations in the LXRresponse element DR4 (direct repeat 4) and the E-box. The
transcriptional regulation of LXR target genes by ORP8 could
involve sequestration of an endogenous sterol activator.
Studies have described altered OSBP/ORP expression in a
variety of disease conditions, but, for the most part, functional
links are absent. A microarray screen for monocyte genes
affected by immune complexes containing oxLDL (oxidized
LDL) identified OSBP as a potential mediator of pro-survival and
inflammatory responses [119]. A miRNA (microRNA) screen in
oxLDL-stimulated monocytes identified an up-regulated ORP9related miRNA that inhibited ORP9 expression, lipid uptake
and inflammatory cytokine secretion [120]. Altered OSBP/
ORP expression occurs in a variety of seemingly unrelated
pathological and experimental conditions: ORP1 is associated
with genomic imprinting clusters in Beckwith–Wiedemann
syndrome [121,122]; expression of ORP8 is a response to
antipsychotic drug treatment [123]; increased ORP6 expression
is observed during during neural lineage choice [124]; and ORP4
has been identified as a blood dissemination marker for tumour
metastasis [125].
SNPs (single nucleotide polymorphisms) in subfamily VI
(ORP10 and 11) have been implicated in dyslipidaemic disease.
ORP11 expression is up-regulated in adipose tissues of men
with a high risk of cardiovascular disease [126]. Further
investigation associated a number of SNPs in the ORP11 gene
with cardiovascular disease risk factors, including hypertension,
LDL-cholesterol plasma levels and hyperglycaemia [127].
Polymorphisms in the ORP10 gene are linked to high TAG serum
levels in Finnish dyslipidaemic subjects [115]. The effect of
these polymorphisms on ORP10 function is unknown, but RNAi
silencing in Huh7 cells resulted in increased TAG and cholesterol
biosynthesis, and secretion of ApoB100 (apolipoprotein B-100)
and ApoA1 in the culture medium. These effects were not
correlated with expression of known lipid metabolic enzymes, but
could be related to organelle and lipid transfer mediated through
interaction with microtubules.
VAP and OSBP have been implicated in the replication of HCV
(hepatitis C virus), a single-strand RNA virus that assembles on
ER membranes and lipid droplets [128]. NS5A (non-structural
protein 5A) and the RNA-dependent RNA polymerase NS5B
(non-structural protein 5B) both interact with VAP-A and VAP-B
on the ER to form a stable replication complex [129–131].
HCV replication in cells was significantly diminished by VAP
silencing [129,132] or hyperphosphorylation of NS5A, which
disrupted its interaction with VAP-A [133]. OSBP also interacts
with NS5A and is required for HCV replication in human
hepatoma cells, an activity that appeared to involve targeting
to the Golgi apparatus [134]. VAP and OSBP interact with
the N-terminus of NS5A, possibly to enhance viral replication
by a scaffolding or membrane-targeting mechanism, or by
facilitating the creation of an optimal membrane environment
for viral assembly. The p48 protein of the single-strand RNAencoded Norwalk virus (Norovirus) also interacts with VAP-A
suggesting it is a commonly used host factor for viral replication
[135].
21
CONCLUSIONS
Altering the expression of OSBP/ORP family members, or measuring gene expression in response to specific cues or agents,
has identified seemingly diverse and sometimes discordant
functions for this large gene family. However, recent evidence
that mammalian and yeast ORPs can transfer sterols in vitro and
in vivo, coupled with their conserved sterol transport-like domain,
suggests that many of the observed functional outputs are the
consequences of this fundamental activity. The major challenge
in upcoming years will be to identify where OSBP/ORPs transfer
sterols, the mechanisms that control directionality, endogenous
ligand(s) and how the transfer is linked to a sterol-sensing
function, either at the protein or membrane level.
FUNDING
The work of our laboratory is supported by the Canadian Institutes for Health Research
[grant number 62916] and a grant-in-aid from the Heart and Stroke Foundation of Canada
(Nova Scotia).
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