oxysterol binding protein homologues : novel functions in
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Minerva Foundation Institute for Medical Research and
National Institute for Health and Welfare and
Department of Medical Biochemistry and Developmental Biology, Institute of Biomedicine, Faculty of Medicine, University of Helsinki
Oxysterol binding protein homologues: Novel functions in intracellular transport
And adipogenesis
YOU ZHOU
ACADEMIC DISSERTATION
To be presented, with the permission of the Faculty of Medicine, University of Helsinki for public examination in Lecture Hall 2, Biomedicum Helsinki
on January 25th, 2013, at 12 noon.
Helsinki 2013
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SUPERVISOR: Adjunct Professor Vesa Olkkonen, PhD Minerva Foundation Institute for Medical Research Biomedicum Helsinki Finland REVIEWERS: Professor Pekka Lappalainen, PhD Institute of Biotechnology University of Helsinki Helsinki Finland Adjunct Professor Pirkko Pussinen, PhD Institute of Dentistry University of Helsinki Helsinki Finland OPPONENT: Adjunct Professor Anna-Liisa Levonen, MD, PhD A.I.Virtanen Institute for Molecular Sciences University of Eastern Finland Kuopio Finland ISBN 978-952-10-8594-9 (paperback) ISBN 978-952-10-8595-6 (pdf) Cover designer: You Zhou Helsinki University Print (Unigrafia) HELSINKI 2013
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To Eunjee and Joel
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Table of Contents List of original publications ........................................................................................ 6 ABSTRACT .................................................................................................................. 7 ABBREVIATIONS ...................................................................................................... 9 1 INTRODUCTION .................................................................................................. 12 2 REVIEW OF THE LITERATURE ...................................................................... 14
2.1 Lipids ................................................................................................................. 14 2.2 Intracellular lipid transport ............................................................................ 16
2.2.1 Vesicular transport ..................................................................................... 16 2.2.2 Non-vesicular transport .............................................................................. 16
2.3 Lipid signaling .................................................................................................. 19 2.4 Oxysterols ......................................................................................................... 20
2.4.1 Oxysterols in Signaling and Development .................................................. 22 2.4.2 Oxysterols mediate regulation of lipid metabolism .................................... 23 2.4.3 Oxysterols induce apoptosis ....................................................................... 26 2.4.4 Oxysterols and metabolic diseases ............................................................. 26 2.4.5 Oxysterols in Niemann-Pick type C disease ............................................... 27
2.5 Cytoplasmic oxysterol binding proteins......................................................... 29 2.5.1 Identification of OSBP and ORPs ............................................................... 29 2.5.2 Structure of ORPs ....................................................................................... 30 2.5.3 Cellular sterol ligands of ORPs .................................................................. 32 2.5.4 Subcellular distribution of ORPs ................................................................ 33 2.5.5 ORPs act as sterol transporters .................................................................. 35 2.5.6 ORPs regulate cellular lipid homeostasis ................................................... 36 2.5.7 The role of ORPs at MCSs (membrane contact sites) ................................. 37 2.5.8 ORPs modulate signaling processes ........................................................... 38 2.5.9 The roles of ORPs in human diseases ......................................................... 39
2.6 GATE-16, GS28 and intracellular trafficking............................................... 41 2.7 Adipocytes and the Simpson-Golabi-Behmel syndrome (SGBS) model ..... 42
3 AIMS OF THE STUDY ......................................................................................... 45 4 Materials and methods ........................................................................................... 46
4.1 List of Published methods ............................................................................... 46 4.2 Brief descriptions of the methods employed .................................................. 46
5 RESULTS AND DISCUSSION ............................................................................. 52 5.1 Characterization of ORP11 ............................................................................. 52
5.1.1 Tissue and cell type specific expression patterns of ORP11 ....................... 52
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5.1.2 Subcellular localization of ORP11 ............................................................. 54 5.2 Cellular roles of ORP11 ................................................................................... 55
5.2.1 Overexpression of ORP11 induces the formation of lamellar lipid bodies 55 5.2.2 ORP11 interacts with ORP9 ....................................................................... 56 5.2.3 ORP11 Golgi association is facilitated by ORP9 ....................................... 57
5.3 ORPs in human adipose depots and cultured adipocytes ............................ 58 5.3.1 OSBP/ORP copy numbers in human adipose depots and SGBS adipocytes............................................................................................................................... 59 5.3.2 OSBP/ORP expression during SGBS adipocyte differentiation ................. 60 5.3.3 Effect of ORP silencing/overexpression on adipocyte differentiation ........ 61
5.4 ORP7 and GATE-16 ........................................................................................ 63 5.4.1 ORP7 interacts with GATE-16 .................................................................... 63 5.4.2 Subcellular localization of ORP7, GATE-16 and GS28 ............................. 64 5.4.3 ORP7 regulates GS28 protein level through transcriptional regulation .... 65 5.4.4 ORP7 affects GS28 degradation on proteasomes via GATE-16 ................ 65 5.4.5 ORP7 potentiates 25-OH-induced degradation of GS28 ............................ 66
6 CONCLUSIONS AND PERSPECTIVES ............................................................ 68 7 ACKNOWLEDGEMENTS ................................................................................... 70 8 REFERENCES ........................................................................................................ 73
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List of original publications This thesis is based on the following original research articles, which are referred to in
the text by their Roman numerals.
I. Y. Zhou, S. Li, M.I. Mäyränpää, W. Zhong, N. Back, D. Yan, V.M. Olkkonen,
OSBP-related protein 11 (ORP11) dimerizes with ORP9 and localizes at the
Golgi-late endosome interface, Exp. Cell Res. 316 (2010) 3304-3316.
II. W. Zhong, Y. Zhou, S. Li, T. Zhou, H. Ma, K. Wei, H. Li, V.M. Olkkonen, D.
Yan, OSBP-related protein 7 interacts with GATE-16 and negatively regulates
GS28 protein stability, Exp. Cell Res. 317 (2011) 2353-2363.
III. Y. Zhou, M.R. Robciuc, M. Wabitsch, A. Juuti, M. Leivonen, C. Ehnholm, H.
Yki-Järvinen, V.M. Olkkonen, OSBP-related proteins (ORPs) in human
adipose depots and cultured adipocytes: Evidence for impacts on the adipocyte
phenotype, PLoS One. 7 (2012) e45352.
The original articles are reproduced by kind permission of the copyright holders.
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ABSTRACT
Lipids, as the major components of cell membranes, are multi-functional molecules
that also critically contribute to signal transduction, transcriptional regulation and
membrane trafficking. The voice from single lipid molecule mixes together to create a
“balancing symphony or chorus” called lipid homeostasis. Disturbances in the
“molecular song” by lipids trigger profound physiological responses, which are
reflected as metabolic disorders, such as obesity, metabolic syndrome, and
atherosclerosis. Hence, it is important to understand the semiotics of this molecular
language.
Lipid homeostasis is context-dependent and regulated coordinately by a variety of
lipids per se and proteins. Oxysterol binding protein (OSBP) and its homologues
(ORPs) are implicated to regulate lipid homeostasis, sterol transfer and cell signaling.
In this thesis, two ORPs, ORP11 and ORP7 have been extensively studied. ORP11 is
abundant in human ovary, testis, kidney, liver, stomach, brain and adipose tissue and
resides at the Golgi-Late endosome interface. ORP11 forms a dimer with its close
homologue, ORP9, the interaction occuring in the region of aa154-292 in ORP11 and
98-372 in ORP9, which maintains the subcellular distribution of ORP11.
ORP7 interacts with GATE-16 and might be involved in autophagosome biogenesis.
Excess ORP7 induces recruitment of GATE-16 from Golgi to autophagosomes. ORP7
thereby regulates the proteosome-dependent degradation of Golgi v-SNARE protein,
GS28, another binding partner of GATE-16. 25-hydroxycholesterol, a ligand of ORP7,
modifies GS28 protein stability, an effect which is influenced by ORP7.
Being the largest endocrine organ in human body, adipose tissue is the primary place
to store fat. However, there is no study to compare expression patterns of ORPs in
white adipose depots and adipose cells. In this thesis, we showed that human
subcutaneous and visceral adipose depots as well as Simpson–Golabi–Behmel
syndrome (SGBS) adipocytes share similar ORP expression patterns, indicating that
the mRNA signals of ORPs in adipose tissues predominantly originate from
adipocytes. During adipogenesis, ORP2, ORP3, ORP4, ORP7 and ORP8 mRNA
levels were downregulated, whereas ORP11 was upregulated. Silencing of ORP11
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resulted in a decreased expression of adiponectin and aP2, while overexpression of
ORP8 down-regulated the aP2 mRNA. Interestingly, silencing of ORP11 and
overexpression of ORP8 significantly impaired triglyceride storage in the adipocytes.
Taken together, the work in this thesis identifies protein binding partners of ORPs and
indicates their potential roles in protein distribution and stability, intracellular
trafficking, sterol sensing, and the adipocyte phenotype.
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ABBREVIATIONS
ABCA1
ACAT
ANK
ApoA-I
ARF
Atg8
BiFC
BMI
BSEP
CAD
CERT
CNS
COP
DAG
DHE
EPOX
ER
ERK
FAPP2
FCH
FFAT
GAPDH
GATE-16
GLTP
HDL
Hh
HMGCoAR
Insig
KC
LC3
LD
ATP-binding cassette transporter A1
acyl-CoA:cholesterol acyltransferase
ankyrin repeat
apolipoprotein A-I
ADP-ribosylation factor
autophagy-related protein 8
bimolecular fluorescence complementation
body mass index
bile salt export pump
coronary artery disease
ceramide transfer protein
central nervous system
coatomer protein
diacylglycerol
dehydroergosterol
epoxycholesterol
endoplasmic reticulum
extracellular signal-activated kinase
four-phosphate-adaptor protein 2
familiar combined hyperlipidemia
two phenylalanines in an acidic tract
glyceraldehyde-3-phosphate dehydrogenase
Golgi-associated ATPase enhancer of 16 kDa
glycolipid transfer protein
high density lipoprotein
Hedgehog
3-hydroxy-3-methylglutaryl coenzyme A reductase
Insulin-induced gene
ketocholesterol
microtubule-associated protein 1 light chain 3
lipid droplet
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LDL
LE
LTP
LXR
MCS
NPC
NSF
NVJ
OHC
OSBP
ORP
ORD
PA
PC
PDK-2
PE
PG
PH
PI
PI3K
PI4P
PIP
PITP
PL
PM
PPAR
PS
qPCR
RILP
ROR
RXR
SCAP
SCP
low-density lipoprotein
late endosome
lipid transfer protein
liver X receptor
membrane contact site
Niemann-Pick type C
N-ethylmaleimide sensitive factor
nucleus-vacuole junction
hydroxycholesterol
oxysterol binding protein
oxyterol binding protein related protein
OSBP-related (ligand binding) domain
phosphatidic acid
phosphatidylcholine
phosphoinositide-dependent kinase-2
phosphatidylethanolamine
phosphatidylglycerol
pleckstrin homology
phosphatidylinositol
phosphatidylinositol 3-kinase
phosphatidylinositol 4-phosphate
phosphoinositide
PI transfer protein
phospholipid
plasma membrane
peroxisome proliferator-activated receptor
phosphatidylserine
quantitative real-time PCR
Rab-interacting lysosomal protein
receptor-related orphan receptor
retinoid X receptor
SREBP cleavage activating protein
sterol carrier protein
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SD
SDHA
SEM
SGBS
shRNA
SM
SNAP
SNARE
SREBP
STAR
START
T2D
TG
TGN
t-SNARE
VAP
VLDL
vesicle SNARE
standard deviation
succinate dehydrogenase subunit alpha
standard error of mean
Simpson–Golabi–Behmel syndrome
short hairpin RNA
sphingomyelin
soluble NSF attachment protein
soluble NSF attachment protein receptors
sterol regulatory element binding protein
steroidogenic acute regulatory protein
STAR related lipid transfer protein
type 2 diabetes
triglyceride
trans-Golgi network
target SNARE
vesicle associated membrane-associated protein
very-low-density lipoprotein
v-SNARE
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1 INTRODUCTION The incidence and prevalence of metabolic disorders have increased drastically during
the recent decades. Owing to their characteristics such as chronicity and the long-term
interventions required, metabolic disorders constitute a serious health threat and pose
a heavy economic burden on societies. These disorders such as obesity, diabetes,
atherosclerosis, high blood pressure and cardiovascular disease are tightly connected
with the metabolism of lipids, molecules playing essential roles in the control of
energy homeostasis, organ physiology and cellular metabolism. Disturbances of lipid
homeostasis can provoke a variety of cellular responses, and further trigger life-
threatening metabolic diseases. It is hence important to understand lipid signaling,
transport and related cellular processes.
Oxysterols are 27-carbon oxygenated derivatives of cholesterol, which are present at
low concentrations in healthy mammalian tissues. However, they are enriched in
atherosclerotic lesions, macrophage foam cells, gall stones, cataracts and specific low-
density lipoprotein (LDL) subfractions. Oxysterols play important roles in signaling
and development through regulation of Hedgehog signaling pathway and estrogen
receptor function. Furthermore, oxysterols mediate lipid metabolism by controlling a
number of transcription factors. In addition, oxysterols associate with a variety of
complex metabolic diseases as well as the monogenic Niemann-Pick type C disease
(NPC).
Oxysterol binding protein (OSBP) was originally identified because of its ability to
bind oxysterols. OSBP and its homologues constitute a large family of lipid transfer
proteins. They are conserved throughout the eukaryotic kingdom and play diverse
functions in a wide variety of cellular processes including lipid metabolism, cell
signaling, vesicular and non-vesicular trafficking. In humans, 12 ORP genes encode
more than 16 different protein products through differential pre-mRNA splicing and
use of alternate promoters. The core lipid binding OSBP-related domain (ORD) at the
C-terminus containing the conserved motif EQVSHHPP is the feature common for all
ORPs. Many ORPs are dual membrane-targeting proteins: This means that they carry
a two phenylalanines in an acidic tract (FFAT) motif which anchors the proteins to
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the ER membrane; a pleckstrin homology (PH) domain at the N-terminus of many
ORPs binds phosphoinositides, thus also contributing to the subcellular localization of
the proteins. This specific characteristic suggests that ORPs have profound roles at
membrane contact sites, at which ER is closely apposed with other organelles. ORPs
regulate cellular lipid homeostasis and signaling and their roles in human disease are
emerging. It is thus extremely interesting and of importance to investigate the
function of ORP in cellular metabolism and in pathways disturbed in human disease.
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2 REVIEW OF THE LITERATURE
2.1 Lipids More than 1,000 chemically distinct lipid species are organized into the separate
membrane-bound compartments or organelles of eukaryotic cells (Sleight. 1987). The
primary cellular role of lipids is to form the bilayer barrier of cells and organelles and
further to contribute to the intrinsic properties of membranes, such as thickness,
permeability, asymmetry and curvature. Thus, different biological membranes vary in
lipid composition. For example, plasma membrane (PM) is enriched in sterols and
sphingolipids and exhibits a transverse lipid asymmetry, whereas the endoplasmic
reticulum (ER), which contains a low level of both of the above lipids, displays a
symmetrical transbilayer lipid distribution (Pomorski, et al. 2001).
Lipids in mammalian cell membranes are divided into three major groups:
glycerolipids, sphingolipids and sterols (Sprong, et al. 2001). Glycerophospolipids,
derived from glycerol-3-phosphate with two fatty acid chains, are the major building
blocks of membranes (Figure 1). Another important group of glycerolipids are the
mono-, di- and triacylglycerols. Glycerophospholipids and sphingomyelin (SM),
collectively designated phospholipids (PLs) such as phosphatidic acid (PA),
phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG),
phosphatidylinositol (PI), and cardiolipin are defined based on their head groups. PLs
are distributed asymmetrically in the PM. For example, PC and SM are mainly found
in the outer leaflet while PE and PS are generally concentrated in the inner leaflet
(van Meer, et al. 2008). In contrast, ER membrane has a symmetric lipid arrangement
with enriched unsaturated glycerophospholipids which maintain the flexible property
of membrane and facilitate the transportation of synthesized proteins.
Sphingolipids (Figure 1) based on a C18 sphingoid base are enriched in the PM, Golgi
and endosomal membranes. Different classes of sphingolipids are defined according
to the structure of their head groups. The head groups phosphocholine and
phosphoethanolamine form SM and ethanolaminephosphorylceramide, respectively.
Alternatively, glucose or galactose in this position produces different forms of
glycosphingolipids (Figure 1) which are abundant in the outer leaflet of the PM and
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luminal leaflet of intracellular vesicles. Glycosphingolipids are capable of mediating
cell-cell interactions and signal transduction (Schnaar. 2004, Lahiri & Futerman.
2007).
Figure 1.
The three main classes of mammalian membrane lipids. C stands for a variety of
polar head group according to species. Abbreviations: PC, phosphatidylcholine; PE,
phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol
(Reproduced with permission, van Meer & de Kroon. 2011). Please see text for
details.
Sterols are present as different forms with comparable functions in most eukaryotic
cells. Cholesterol is found in mammals, sitosterol in plants and ergosterol in fungi.
Based on a planar four-ring structure (Figure 1), these sterols have similar physical
properties and intracellular distribution (Bagnat, et al. 2000, Bhat & Panstruga. 2005,
Prinz. 2007). Cholesterol shows a gradient distribution across the secretory system,
with the lowest concentrations in the ER and the highest in the PM. Cholesterol is
necessary for the viability of mammalian cells and maintenance of the permeability
barrier function of the PM (Prinz. 2007).
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2.2 Intracellular lipid transport Because the concentrations of lipids differ largely in various subcellular organelles,
intracellular lipid trafficking has been extensively investigated. However, how the
lipids are transported to their target destinations remains not fully understood.
Increasing lines of evidence suggest that lipids are delivered to their destinations
through both vesicular and non-vesicular pathways (Funato & Riezman. 2001, Lev.
2010, Lev. 2012).
2.2.1 Vesicular transport
As the primary constituent of vesicles, large amounts of lipids are transported by
vesicles between the various subcellular compartments. Transport of sphingolipids
and glycerolipids is to a large extent based on vesicular transport. To maintain the
high concentration of cholesterol in the PM, cholesterol is incorporated into vesicles
destined to the PM. However, a major part of cholesterol is known to bypass the
vesicle transport through the Golgi apparatus (Heino, et al. 2000). Sphingomyelin and
sterols are partially segregated from Coatomer protein (COP)I-coated vesicles. This
segregation process is important for the intra-Golgi transport and retrograde transport
from Golgi to ER (Prinz. 2007, Holthuis & Levine. 2005). In addition, the affinity to
the microdomains and the extent of membrane curvature are suggested to drive the
sorting of lipids (Schmitz & Grandl. 2009a). The key regulators involved in vesicular
trafficking are Rab GTPases that provide compartmental specificity for membrane
trafficking (Fukuda. 2008), while vesicle fusion is mediated by soluble N-
ethylmaleimide sensitive factor attachment protein receptors (SNAREs) (Pfeffer.
1996).
2.2.2 Non-vesicular transport
Although vast majority of lipid trafficking occurs between organelles participating in
vesicle transport, substantial lipid exchange has been detected between compartments
which are not directly connected by the vesicular transport machinery. Such are the
ER and the PM, the ER and the trans-Golgi, the ER and endosomes, the ER and lipid
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droplets, as well as the ER and mitochondria (Hanada, et al. 2003, Raychaudhuri, et al.
2006). Lipid transport has also been seen under conditions in which vesicular
transport was abrogated by either ATP depletion, temperature reduction or treatment
with specific pharmacological inhibitors (such as brefeldin A and colchicine) (Lev.
2010, Kaplan & Simoni. 1985, Vance, et al. 1991). These findings suggest that non-
vesicular transport plays an important role in intracellular lipid trafficking (e.g.
Ceramide transfer protein (CERT), Funato & Riezman. 2001).
Three mechanisms, monomeric lipid exchange, lateral diffusion and transbilayer flip-
flop are involved in non-vesicular lipid transport (Lev. 2010). Monomeric lipid
exchange is thought to represent a major route for the transport of lipids between
membranes, and is promoted by close apposition of the two membranes. Lipid
molecules are moved across an aqueous phase from the outer leaflet of the donor
membrane to the outer leaflet of the acceptor membrane without the requirement for
metabolic energy. This process could be either spontaneous or assisted by lipid
transfer proteins (LTPs). Lateral diffusion mediates lipid transport in the lateral plane
of the bilayer, which majorly occurs within membranes. Transbilayer flip-flop occurs
spontaneously or is mediated by proteins such as flippases and translocases. The flip-
flop of lipids between the inner and outer leaflets of the membrane bilayer would
further ensure monomeric lipid exchange.
Intracellular movement of lipids is greatly facilitated by the membrane contact sites
(MCSs) via a non-vesicular transport mechanism (Lev. 2010). MCSs are small
cytosolic gaps (10-30 nm) between the membranes of the ER and most other
organelles including the PM, Golgi apparatus, lipid droplets, late endosomes,
lysosomes and mitochondria. These dynamic structures have been identified in all
eukaryotes and are typically enriched in proteins involved in lipid synthesis and
transport or channels responsible for ion transport (Gillon, et al. 2012). MCSs are
formed by the tethering of adjacent membranes via protein-protein or protein-lipid
interactions. Such tethers regulate lipid composition of the tethered membranes and
affect calcium homeostasis (Levine & Loewen. 2006).
In principle, non-vesicular lipid transport could occur spontaneously by lipid
desorption. However, the rate of this process is slow and not sufficient to support
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substantial transport of most lipids. Wirtz and Zilversmit (1968) et al.
initially discovered that lipid transfer proteins (LTPs) are able to accelerate exchange
of different lipid species between membranes in vitro. Since then more and more lipid
transfer proteins have been characterized according to their lipid-transfer domain
(D'Angelo, et al. 2008). In addition, LTPs have been found throughout the eukaryotic
kingdom and have been subdivided into different families on the basis of their
sequence and structural similarity, such as SEC14, PI transfer protein (PITP),
steroidogenic acute regulatory protein (STAR)-related lipid transfer (START)
proteins, glycolipid-transfer protein (GLTP), sterol carrier protein 2/non-specific lipid
transfer protein (SCP2/NSLTP), oxysterol-binding protein (OSBP) and its
homologues (D'Angelo, et al. 2008). Commonly, they have a lipid-binding/transfer
domain and most of them have additional domains with different functions,
responsible for e.g. membrane targeting.
LTPs are present in two distinct conformations: a “closed” or a transport-competent
confirmation in which a hinged lid domain covers the lipid accommodating tunnel,
and an “open” confirmation (Prinz. 2007). The interaction between an LTP and a
membrane is thought to stimulate the transformation between the two conformations.
The stability of the closed lid is secured by hydrophobic interactions. The tunnel
opening is induced by polar interactions between the LTP and membrane
phospholipids. LTPs interact with a donor membrane, open the lipid-binding tunnel,
absorb a lipid, dissociate from the donor membrane, and are found in the close
conformation in the aqueous phase. At the destination on an acceptor membrane, the
lipid molecule is desorbed or exchanged with another lipid while the tunnel is opening.
The whole sequential process facilitates inter-organelle lipid transport and maintains
the overall lipid homeostasis. In addition, some LTPs could bind both acceptor and
donor membranes simultaneously (Prinz. 2007). Moreover, it is of interest to note that
the membrane binding motifs of LTPs are thought to affect the transport of specific
lipids through binding to other lipids/proteins on the membranes.
LTPs show different binding affinities to the headgroup and backbone of lipid
substrates (Kumagai, et al. 2005). Based on their lipid-binding specificity and
transfer capability, LTPs can be categorized in three major classes: phospholipid-,
sterol- and sphingolipid-transfer proteins. Phospholipid-transfer proteins in mammals
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represent three major classes: PC-transfer protein (PCTP), PI-transfer protein (PITP)
and nonspecific LTP (NSLTP), which is, however, also capable of sterol transfer and
has thus also been called sterol carrier protein 2 (SCP2) (Prinz. 2007). OSBP and
OSBP-related proteins (ORP), certain members of START family (e.g.,
STARD3/MLN64), Niemann-Pick type C (NPC) 2 are considered to be sterol-transfer
proteins. Ceramide transfer protein (CERT) and four-phosphate-adaptor protein 2
(FAPP2), which transfer ceramide and glucosylceramide respectively,and glycolipid
transfer protein (GLTP) are examples of sphingolipid-transfer proteins (Yamaji, et al.
2008, Malinina, et al. 2004).
A number of LTPs have been detected at MCSs with diverse functions. For example,
oxyterol binding protein related protein ORP1L was reported to induce and maintain
the formation of ER-late endosome MCSs under low cholesterol conditions (Rocha, et
al. 2009, Vihervaara, et al. 2011b). CERT, which shuttles ceramide between the ER
and the trans-Golgi, could specifically target the ER-Golgi MCSs for transportation. It
has two targeting domains – that is a pleckstrin homology (PH) domain which binds
phosphatidylinositol 4-phosphate (PI4P) and a FFAT motif that interacts with the ER
resident protein of vesicle associated membrane-associated proteins (VAPs) family
(Hanada, et al. 2003).
2.3 Lipid signaling
Lipids have been recognized as signaling molecules and their levels contribute to
crucial functional consequences. A major discovery in the 1980s was that
diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (Ins(1,4,5)P3), the products of
phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2 or PtdIns(4,5)P2),were identified as
second messengers to provoke the activation of protein kinase C (PKC) and calcium
release into the cytosol (Nishizuka. 1992). Over the past decades, the eicosanoids
produced from arachidonic acid have also been identified as the key signaling lipids
that have the capacity to control inflammatory responses (Serhan & Savill. 2005).
Specific eicosanoids apparently bind to peroxisome proliferator-activated receptors
(PPARs), which further modulate lipid homeostasis (Chawla, et al. 2001). Further
studies have reported that phosphatidylinositol-3,4,5-trisphosphate, a minor product
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of inositol phospholipid metabolism, serves as a signaling lipid that mediates cell
growth, proliferation and motility (Engelman, et al. 2006).
Sphingolipids are an important group of bioactive lipids. Of these, sphingosine is the
first one to be identified. The phosphorylaton of sphingosine forms sphingosine-1-
phosphate that exerts important functions in cell survival, growth, migration, and
inflammation (Hannun & Obeid. 2008). Ceramide plays crucial roles in cell
differentiation, senescence and apoptosis. It can be generated rapidly from
sphingomyelin in response to the activation of sphingomyelinase. Under different
cellular stress conditions, such as DNA damage or lysosome disruption, ceramide
concentrations increase. Moreover, ceramide has the capacity to regulate insulin
signaling in regulating several crucial intermediates in the insulin signaling pathway
(Summers. 2006).
Disturbances of the lipid signaling pathways exert pleiotropic effects on inflammatory
processes and contribute to a variety of diseases such as cancer, atherosclerosis,
hypertension, type 2 diabetes and cardiovascular diseases. In addition, increased lipid
signaling is suggested to play a role in the development of tumors from benign to
malignant stage (Wymann & Schneiter. 2008).
2.4 Oxysterols
Oxysterols are 27-carbon oxygenated products of cholesterol that arise through
enzymatic or non-enzymatic oxidation processes, or are absorbed from the diet (Vaya,
et al. 2011). Several major oxysterols are produced as intermediates in the
biosynthesis of bile acids or steroid hormones. The common modifications of
cholesterol occurring in oxysterols are hydroxyl, keto, hydroperoxy, epoxy and
carboxyl moieties. The most abundant oxysterols in human serum are 24(S)-, 27-, 7α-,
and 4β-hydroxycholesterol (OHC) (Figure 2), which are generated in reactions
catalyzed by mitochondrial or ER cholesterol hydroxylases belonging to the
cytochrome P450 family (Olkkonen. 2009) . 24(S)-OHC is exclusively present in
neurons of the central nervous system (CNS). The synthesis of 24(S)-OHC catalyzed
by the cholesterol hydroxylase CYP46A1 maintains the CNS sterol homeostasis
21
(Bjorkhem. 2007). 27-OHC, which is generated in the liver, catalyzed by CYP27A1,
serves as an intermediate of the alternative, so called acidic bile acid synthetic
pathway (Bjorkhem & Eggertsen. 2001). CYP27A1, is however also expressed by
other, non-hepatic cells (Babiker, et al. 1997). Similarly, 7α-OHC is an intermediate
in the neutral pathway of the bile acid synthesis. Thus, 7α-OHC is primarily produced
in the liver catalyzed by CYP7A1. 4β-OHC is converted from cholesterol by the drug
metabolizing enzyme CYP3A4 which is induced by certain anti-epileptic
pharmaceuticals (Bjorkhem & Eggertsen. 2001).
Oxysterols can also be synthesized through non-enzymatic, free radical or lipid
peroxide processes, which are often designated cholesterol autoxidation. For example,
1% of the sterols in the western diet has been estimated to be non-enzymatically
oxidized (van de Bovenkamp, et al. 1988). The autoxidation commonly occurs at the
7-position of the cholesterol B-ring, which yields 7-ketocholesterol (7-KC), 7α-OHC
or 7β-OHC (Figure 2).
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Figure 2. Structures of the selected common oxysterols. Oxygen-containing
modifications at the different positions of cholesterol produce a variety of oxysterols.
2.4.1 Oxysterols in Signaling and Development
2.4.1.1 Oxysterols regulate Hedgehog signaling pathway The Hedgehog (Hh) signaling pathway plays a fundamental role in embryonic
development, physiological processes and stem-cell renewal. Cororan and Scott first
reported that cholesterol or certain oxysterols are critically required for Sonic Hh
pathway signal transduction (Corcoran & Scott. 2006). Dwyer et al. demonstrated that
20(S)- and 22(S)-OHC induce the osteoinductive effects through the activation of Hh
signaling in pluripotent mesenchymal cells (Dwyer, et al. 2007). Furthermore,
Parhami's group found that 22R-OHC, 20(S)-OHC and its analogues exert effects of
inhibiting adipocyte differentiation through the Hh signaling pathway (Kha, et al.
2004, Kim, et al. 2007, Johnson, et al. 2011). Very recently, Nachtergaele et al.
presented the evidence that the enantiomer and an epimer of 20(S)-OHC regulate Hh
23
signaling pathway through activation of a specific target protein Smoothened
(Nachtergaele, et al. 2012). These lines of evidence established a novel role of
oxysterols as developmental regulators.
2.4.1.2 27-OHC regulates estrogen receptor function
Two different forms of the estrogen receptor, ER-α and ER-β, are members of nuclear
hormone superfamily that mediate a number of physiological processes. One
important role of estrogen receptors is the regulation of cardiovascular system where
estrogens exert protective effects on the blood-vessel wall (Mendelsohn & Karas.
1999). Umetani et al. reported that 27-OHC directly antagonizes the transcriptional
and non-transcriptional functions of the estrogen receptors, resulting in a loss of the
cardioprotective function of estrogen (Umetani, et al. 2007). Furthermore, they
demonstrated that 27-OHC acts as an endogenous selective estrogen receptor
modulator based on its cell-type specific proestrogenic action.
2.4.2 Oxysterols mediate regulation of lipid metabolism
2.4.2.1 Oxysterols act as the ligands of Liver X receptor
There are two isoforms of nuclear receptors called liver X receptors (LXRs) in
mammals, LXRα and LXRβ. LXRα is abundant in liver, intestine, spleen, kidney,
macrophages and adipose tissue, while LXRβ is ubiquitously expressed at a low level
(Beltowski. 2008). LXRs form heterodimers with retinoid X receptor (RXR), a
common partner for several nuclear receptors such as peroxisome proliferator
activated receptors (PPARs) and vitamin D receptor. The formed complex can be
activated through liganding of either partner to regulate gene expression in response
to the recruitment of coactivators or corepressors. The activation further regulates the
intestinal cholesterol absorption, reverse cholesterol transport, bile acid synthesis,
cholesterol synthesis, hepatic lipogenesis, high density lipoprotein (HDL) formation
and inflammatory conditions (Beltowski. 2008). In addition, several studies have
reported that LXR agonists reduce the size of atherosclerotic lesions in mouse models
(Joseph, et al. 2002, Terasaka, et al. 2003). This effective protection is valid not only
24
during the development of lesions but also in the established atherosclerosis (Levin, et
al. 2005). Increasing lines of evidence clearly show that LXRs indeed execute an anti-
atherogenic function and their agonists are thus potential drugs for atherosclerosis.
Janowski et al. first demonstrated that oxysterols are physiological activating ligands
for LXRs (Janowski, et al. 1996). Since then, the interest towards oxysterols has
greatly intensified. It has been reported that 24(S)-OHC, 22(R)-OHC, 20(S)-OHC,
27-OHC and 24(S),25-epoxycholesterol (EPOX), are the common endogenous LXR
agonists (Janowski, et al. 1996, Lehmann, et al. 1997, Fu, et al. 2001). Triple-
knockout mice lacking CYP46A1, CYP27A1 and cholesterol 25-hydroxylase
responsible for the biosynthesis of three oxysterols, 24S-OHC, 25-OHC, and 27-OHC,
showed impaired responses of LXR target genes to dietary cholesterol, confirming the
view that endogenous oxysterols have an essential role in LXR activation (Chen, et al.
2007). However, it should be noted that not all oxysterols are LXR ligands. Most non-
enzymatically produced oxysterols such as 7α-OHC and 7β-OHC are not ligands for
the LXRs (Janowski, et al. 1999).
2.4.2.2 Oxysterols regulate sterol regulatory element binding protein pathway
Sterol regulatory element binding proteins (SREBPs), designated as SREBP-1a,
SREBP-1c, and SREBP-2, are transcriptional factors and control fatty acid and
cholesterol synthesis. SREBPs are synthesized in the ER as membrane-bound
precursors and form complexes with another membrane protein, SREBP cleavage
activating protein (SCAP). SCAP is considered to be a cholesterol-sensor protein
which controls the intracellular localization and proteolytic maturation of SREBPs. In
the conditions of low sterol levels, SREBP-SCAP are delivered to the Golgi apparatus
by COPII vesicles, where SREBP are proteolytically cleaved to release a soluble N-
terminal domain that is translocated to the nucleus and binds to specific sterol
regulatory element DNA sequences TCACNCCAC in the promoter regions of target
genes. This binding triggers the expression of encoded proteins such as LDL-receptor
and enzymes involved in sterol synthesis. When sterol levels are sufficient in the cells,
the sterols induce SCAP to bind to ER anchor proteins called Insig (Insulin-induced
gene), and COPII can no longer bind to SCAP. Hence, SREBP are inhibited from
25
moving to Golgi and are retained in the ER. Although cholesterol and oxysterols both
stimulate the interaction between SCAP and Insig proteins, they operate in two
different, converging mechanisms. Cholesterol binds to SCAP whereas 25-OHC
directly binds to the Insig proteins, eliciting their conformational change, which
increases the binding affinity of SCAP-Insig complexes and prevents SREBP exit
from the ER (Adams, et al. 2004). Oxysterols associated with Insig also induce the
degradation and ubiquitination of 3-hydroxy-3-methylglutaryl coenzyme A reductase
(HMGCoAR), a key rate limiting enzyme of the sterol biosynthetic pathway, in a
sterol-dependent manner. 24(S)-25 EPOX, a side product of cholesterol biosynthesis
(Figure 2), works as a potent warning system to acutely regulate cholesterol synthesis
by suppressing SREBP2 processing and HMGCoAR activity (Janowski, et al. 2001,
Wong, et al. 2007).
2.4.2.3 Oxysterols regulate other transcriptional factors
As discussed in chapter 2.4.2.1, it is well established that oxysterols act as agonists
for LXRs. Moreover, oxysterols have been reported as the ligands for certain other
transcriptional factors. Several specific oxysterols could increase steroidogenic factor-
1 (SF-1)-dependent steroidogenic acute regulatory protein (StAR) promoter activity,
as well as promoters of P450 enzymes in the specific cell types (Reyland, et al. 2000).
StAR enhances the delivery of cholesterol to the mitochondrial membrane and
regulates the synthesis of steroid hormones in steroidogenic tissues.
It is widely accepted that bile salt export pump (BSEP) is responsible for the bile acid
secretion. 22(R)-OHC has been reported to markedly induce BSEP expression
through binding to the nuclear receptor farnesoid X receptor (FXR) instead of LXR
(Deng, et al. 2006).
The retinoid acid receptor-related orphan receptors (RORs), including the α, β, γ
isoforms, are important nuclear receptors: RORα regulates cerebellum development,
bone formation and immune response while RORγ plays an essential role in the
development of lymphoid tissues and T cells. Both of these receptors are able to
operate as regulators of circadian rhythms (Kang, et al. 2007). Wang et al. found that
26
the transcriptional activity of RORα and RORγ could be suppressed by direct binding
of the antagonists 7α-OHC or 24(S)-OHC, respectively (Wang, et al. 2010b, Wang, et
al. 2010a). Moreover, they found that 24(R)-OHC and 24(S),25-EPOX both regulated
RORγ activity selectively. These findings suggest that RORα and RORγ may serve as
sensors of oxysterols and have overlapping ligand preference and functional
cooperation with LXR (Wang, et al. 2010a).
2.4.3 Oxysterols induce apoptosis
Apoptosis is a genetically controlled process of programmed cell death characterized
by distinct morphological characteristics and energy-dependent mechanisms.
Normally, apoptosis occurs during embryonic development and aging. It is well
accepted as a homeostatic mechanism to maintain cell populations in tissues and a
defense mechanism in immune responses (Elmore. 2007). The apoptosis process
includes the activation of a family of cysteine proteases called “caspases” and a
complex of events related to the cell death in response to a wide variety of stimuli,
irradiation, hormones and oxysterols (Elmore. 2007).
Oxysterols modify the biophysical and biochemical properties of membranes and
have the capacity to induce cell death through two major apoptotic pathways, the
mitochondrial (intrinsic) pathway and the receptor-dependent (extrinsic) pathway.
Notably, the apoptotic effects and employed pathways of apoptosis depend on the
particular oxysterol and the cell type. Hence, it is most likely that there is no universal
mechanism of apoptosis induced by oxysterols (Lukyanenko & Lukyanenko. 2009,
Olkkonen, et al. 2012a). Because of the cytotoxic and pro-apoptotic functions,
oxysterols are suggested to be involved in the progress of diseases such as
atherosclerosis and biliary tract diseases (Shibata & Glass. 2010, Wang & Afdhal.
2001).
2.4.4 Oxysterols and metabolic diseases
A number of studies suggest that the plasma concentrations of oxysterols could be
used as indicators of oxidative stress in vivo. Oxysterols have been detected to be
27
remarkably accumulated in LDL fractions of coronary artery disease (CAD) patients
compared to the controls (Olkkonen. 2009). Moreover, it was found that plasma 7-KC
and 7β-OHC were significantly higher in patients with stable CAD compared to
controls with normal coronary arteries (Rimner, et al. 2005). It was reported that the
levels of 7-KC and 7β-OHC were remarkably elevated in the plasma of familiar
combined hyperlipidemia (FCH) patients compared to sex- and age-matched healthy
controls (Arca, et al. 2007). 7-KC concentration in plasma was found to be positively
associated with risk of type 2 diabetes (T2D) mellitus, especially in the group with
multiple coronary risk factors (Endo, et al. 2008). The plasma concentration of 7β-
hydroxycholesterol was reported higher in the patients with T2D than those without
T2D, or with impaired glucose tolerance (Ferderbar, et al. 2007). 27-OHC is regarded
as the most abundant oxysterol in atherosclerotic lesions, the concentration of which
is associated with the severity of atherosclerosis (Garcia-Cruset, et al. 2001).
Several oxysterols have been proposed as disease markers. However, reliable
quantification of oxysterol levels in clinical samples depends on sample freshness,
storage method, assay technique and sample cholesterol levels (Lukyanenko &
Lukyanenko. 2009). Notably, it is not reliable to quantify the levels of non-
enzymatically formed oxysterols in clinical samples because they easily arise
artefactually (Gill, et al. 2008). For example, concerning the measurement of 7-KC in
the cerebrospinal fluid from patients, the results from two groups varied by more than
three orders of magnitude (Diestel, et al. 2003, Leoni, et al. 2005). Hence, it is
important to carefully optimize the methodology employed to quantify the levels of
oxysterols in clinical samples.
2.4.5 Oxysterols in Niemann-Pick type C disease
Niemann-Pick type C (NPC) disease is a rare autosomal recessive neurodegeneration
disorder characterized by accumulation of cholesterol and other lipids in late
endosomal/lysosomal compartments (Vazquez, et al. 2012). Approximately 95% of
NPC cases are caused by mutations in the NPC1 gene while the remaining 5% are
caused by NPC2 gene. The NPC1 gene encodes a membrane protein that localizes in
the late endosomal compartment. The NPC1 protein has 13 trans-membrane domains
28
that have homology to HMGCoAR and SCAP. The NPC2 gene encodes a soluble
lysosomal protein that binds cholesterol. In the late endosomal/lysosomal
compartments, NPC2 delivers LDL-derived cholesterol to the N-terminal domain of
NPC1, which facilitates the insertion of cholesterol to the outer lysosome membrane
(Kwon, et al. 2009). Subsequently, NPC1 mediates the exit of cholesterol from
lysosomes, a process in which the OSBP homologue ORP5 is suggested to play an
important role (Du, et al. 2011).
The markedly reduced ability to esterify LDL-derived cholesterol is a defining
characteristic of NPC disease. Remarkably, the production of oxysterols is impaired
in the NPC disease cells, which might be accounted by the disturbances of cholesterol
homeostasis (Frolov, et al. 2003). In addition, Xie et al. (2003) observed decreased
production of 24(S)-OHC in the NPC1 knockout mice. Furthermore, Infante and
colleagues found that 24(S)-OHC, 25-OHC and 27-OHC bind to the N-terminal
luminal loop of NPC1, suggesting a direct functional cross-talk between oxysterols
and NPC1, which may represent a regulatory ligand interaction (Infante, et al. 2008).
Moreover, oxysterols have been suggested to play roles in the pathology of other
degenerative disorders such as Alzheimer’s disease and age-related macular
degeneration (Jeitner, et al. 2011, Javitt & Javitt. 2009).
29
2.5 Cytoplasmic oxysterol binding proteins A number of LTPs are suggested to transfer sterols between cellular compartments
through non-vesicular pathways. As a large family of LTPs, the cytoplasmic oxysterol
binding protein, OSBP, and its homologues are conserved in the eukaryotic kingdom
from yeast to humans (Olkkonen & Levine. 2004, Olkkonen. 2009). In mammals, 12
ORP genes encode a large number of proteins designated as OSBP-related (ORP) or
OSBP-like (OSBPL) proteins. The ORPs have been implicated in many cellular
processes including lipid metabolism, cell signaling, vesicular trafficking and non-
vesicular sterol transfer. Most ORPs genes are expressed ubiquitously. However, the
distinct tissues and cell types display marked quantitative differences in the
expression pattern of ORP variants (Johansson, et al. 2003, Lehto, et al. 2004).
2.5.1 Identification of OSBP and ORPs
Four decades ago, scientists found that oxysterols were much more potent than
cholesterol itself in suppressing the activity of HMGCoAR, thus thought to act as
feedback signals for the inhibition of cholesterol synthesis (Brown & Goldstein. 1974,
Kandutsch & Chen. 1974, Kandutsch, et al. 1978). These findings prompted a search
for oxysterol responsive proteins that led to the identification of OSBP by Taylor and
co-workers (Taylor, et al. 1984, Taylor & Kandutsch. 1985). Subsequently, OSBP
protein was purified and cDNAs were cloned from rabbit and human (Dawson, et al.
1989a, Dawson, et al. 1989b). OSBP was identified as a cytosolic protein which binds
to oxysterols with high affinity (Levanon, et al. 1990). Treatment of cells with 25-
OHC resulted in the translocation of OSBP from a cytosolic or vesicular compartment
to Golgi membranes rather than the nucleus, indicating that OSBP did not regulate
transcription in a direct manner (Ridgway, et al. 1992).
The cloning of OSBP subsequently led to the identification of its homologues from a
spectrum of eukaryotic organisms. To date, ORPs have been most studied in
mammals and the yeast Saccharomyces cerevisiae. In addition, OSBP and ORPs have
been investigated in Drosophila melanogaster (Alphey, et al. 1998), Caenarhabditis
elegans (Sugawara, et al. 2001, Kobuna, et al. 2010), Dictyostelium discoideum
30
(Fukuzawa & Williams. 2002), Cryptosporidium parvum (Zeng & Zhu. 2006) and
plants (Avrova, et al. 2004, Li, et al. 2008). The existence of ORPs throughout the
eukaryotic kingdom reveals a fundamental and important role of the ORPs family in
eukaryotic evolution.
2.5.2 Structure of ORPs
In humans, 12 ORP genes encode more than 16 different protein products through the
use of different promoters and via differential pre-mRNA splicing. The proteins can
be divided into 6 subfamilies according to the sequence homology (Figure 3). The
mouse genome contains 12 ORP genes with high homology to human ORPs. The
yeast S. cerevisiae genome encodes 7 OSBP homologues called Osh1p-7p, the
pioneering studies of which have provided the evidence to deeply understand the ORP
structure and functions (Im, et al. 2005, Georgiev, et al. 2011, de Saint-Jean, et al.
2011). The short yeast ORP, Osh4p, carrying 435 amino acids, is the only ORP the
structure of which has been solved at atomic resolution (Im, et al. 2005). Structure of
Osh4p was determined in complex with cholesterol, several oxysterols and ergosterol,
the main sterol in yeast. All these virtually identical structures revealed that the
protein has an almost complete β-barrel formed by 19 anti-parallel β sheets, which
operates as a lipid binding pocket or tunnel to accommodate sterols. Moreover, the
structure contains a flexible lid domain that shields the bound sterol from the solvent.
The interaction between sterol side chains and the hydrophobic inner surface of the lid
stabilizes the closed confirmation. Notably, the ligand interactions are substantially
flexible because of the mediating effects by water molecules within the pocket,
explaining the ability of the protein to bind a wide range of sterols. Sterols could enter
and exit the binding pocket when the lid is open, which may be triggered by the
interaction with membranes. Hence, the conformational change in the protein is
stimulated upon binding and release of sterols. In addition, structures of the OSBP-
related ligand binding domains (ORDs) of mammalian ORPs have been modeled on
the basis of the Osh4p structure (Vihervaara, et al. 2011b, Suchanek, et al. 2007). This
structure suggests that ORPs might act as sterol transporters or mediators of sterol
signals.
31
The common feature for all ORPs is the core lipid binding ORD domain at the C-
terminus, which contains the conserved OSBP signature motif EQVSHHPP. A
majority of ORPs in mammals, and some in yeast, carry long N-terminal extensions
containing a pleckstrin homology (PH) domain. The PH domains of ORPs have been
found to bind phosphoinositides (PIPs) (Johansson, et al. 2005, Lemmon. 2007,
Nissila, et al. 2012), which control the subcellular localization of proteins. The
proteins containing only the ORP domain are called “short ORPs”, while those
carrying a PH domain are designated “long ORPs”. Moreover, many ORPs also carry
a two phenylalanines in an acidic tract (FFAT) motif in the region between the PH
domain and ORD, which commonly exists in the proteins involved in lipid
metabolism and is found to bind by ER-resident proteins called VAPs. Hence, some
ORPs, with both PH and FFAT domains, act as dual membrane-targeting proteins.
However, not all ORPs have these domains: ORP1S and ORP4S lack both, ORP2 and
ORP9S have FFAT motifs but no PH domain, whereas ORP10 and ORP11 lack
FFAT motifs but have PH domains. Moreover, some ORPs have ankyrin repeats
(ANK) in their N-terminal regions, which are thought to mediate protein-protein
interactions (Figure 3).
32
Figure 3.
A schematic presentation of mammalian ORP protein structures. The roman
numerals on the right side indicate six subfamilies I-VI of ORP proteins. The color
codes are: green, ORP domain, OSBP-related (ligand binding) domain; yellow,
OSBP-fingerprint (OF) motif; red, pleckstrin homology (PH) domain; purple, ankyrin
repeats; black, FFAT motif, two phenylalanines in an acidic tract targeting VAP of
the ER; turquoise, transmembrane domain (TM).
2.5.3 Cellular sterol ligands of ORPs
OSBP was originally identified because of its ability to bind oxysterols (Dawson, et al.
1989b). Subsequently, OSBP was found to bind cholesterol as well (Wang, et al.
2005). The modeling of mammalian ORP structures using Osh4p suggests that they
all have the ability to bind sterols. Using photo-cross-linkable sterol derivatives in live
cells, Suchanek and coworkers found that most of the mammalian ORPs may bind
33
sterols (Suchanek, et al. 2007). The results have been confirmed in in vitro
experiments by using sterol binding assays for ORP1 (Yan, et al. 2007a), ORP2
(Hynynen, et al. 2009), ORP4 (Moreira, et al. 2001, Wang, et al. 2002), ORP8 (Yan,
et al. 2008), ORP9 (Ngo & Ridgway. 2009) and ORP10 (Nissilä, et al. 2012).
However, the binding affinity to individual sterols varies among the ORPs. OSBP and
ORP4 have been found to show higher affinity to oxysterols than cholesterol (Wang,
et al. 2008, Wyles, et al. 2007). Importantly, all ORPs appear to be able to bind
cholesterol. For instance, ORP9 and its close relative ORP10 were recently found to
bind cholesterol but not oxysterols (Nissilä, et al. 2012, Ngo & Ridgway. 2009).
Fragments of OSBP have been found to retain ability to bind sterol, indicating that the
intact ORD of OSBP may not in all cases be required for sterol binding (Wang, et al.
2008). Indeed, the authors suggest that the lid domain of OSBP is not only the region
which controls the entrance and exit of sterols but also acts as the primary sterol-
binding site. The sterol might first anchor to the lid domain and subsequently be
moved into the binding pocket. Moreover, the authors found that a glycine/alanine
rich region near the N-terminus of OSBP, together with PH domain, appears to
regulate cholesterol binding (Wang, et al. 2008).
2.5.4 Subcellular distribution of ORPs The subcellular localization is important to identify the cellular functions of ORPs.
Binding of specific PIPs by PH domains of ORPs, in some cases, contributes to the
localization of the proteins in the cells (Levine & Munro. 2002). For instance, the
localization of OSBP in the trans-Golgi network (TGN) is regulated by the interaction
between its PH domain and PI4P. Addition of 25-OHC to the cells induces the
translocation of OSBP from cytosol to TGN possibly through a ligand-activated
conformational change that exposes the PH domain (Ridgway, et al. 1992). The TGN
localization of OSBP also depends on ADP-ribosylation factor (ARF) 1, a small
GTPase (Levine & Munro. 2002). Moreover, targeting of yeast Osh1p to Golgi occurs
in a similar manner (Roy & Levine. 2004). Very recently, Ridgway and coworkers
found that phosphorylation on two serine-rich motifs specifically controls the
localization of OSBP in the ER via interaction with VAP (Goto, et al. 2012). The PH
34
domains of certain other “long” ORPs such as ORP1L (Johansson, et al. 2003,
Johansson, et al. 2005), ORP3, ORP6, ORP7 (Lehto, et al. 2004) and ORP9 (Ngo &
Ridgway. 2009, Wyles & Ridgway. 2004) have prominent influences on their
localizations.
Most mammalian ORPs, OSBP, ORP1, 2, 3, 4, 6, 7, and 9 contain a FFAT motif that
binds VAPs, ER-resident integral membrane proteins. In yeast, Osh1p, Osh2p and
Osh3p, contain a FFAT-motif. They bind suppressor of choline sensitivity 2 (Scs2p),
a primary VAP homolog in the yeast which is regulated by the phospholipid
composition in the ER (Loewen & Levine. 2005). ORP5 and ORP8, lacking a FFAT
motif, anchor to the ER by a C-terminal transmembrane segment (Ngo, et al. 2010).
Mammalian ORP1L and S. cerevisiae Osh1p and Osh2p, have motifs with ankyrin
repeats (ANK) in their N-terminal regions, which are involved in protein-protein
interactions (Li, et al. 2006). The ANK region of ORP1L binds to the GTP-bound
active form of Rab7, a late endosomal (LE) small GTPase. The interaction contributes
to the localization of ORP1L to a MCS between the ER and LE. Under low
cholesterol conditions, a conformational change of ORP1L allows its ANK region and
FFAT motif to bind Rab7 and VAP protein, respectively, at the same time, which
induces the formation of contact sites between the ER and the LE (Rocha, et al. 2009).
In analogy with ORP1L, OSBP and ORP9 may localize to the MCSs between the ER
and the Golgi (Peretti, et al. 2008, Ngo & Ridgway. 2009, Raychaudhuri & Prinz.
2010).
The multiple targeting domains found in most ORPs facilitate them to shuttle between
two different compartments or to be enriched in MCS regions. In addition, binding of
the FFAT motif and the PH domain to ER and non-ER membrane compartments
simultaneously might facilitate the formation of close contact sites between the ER
and a second organelle (Olkkonen & Levine. 2004, Raychaudhuri & Prinz. 2010).
Increasing lines of evidence suggest that ORPs might operate as LTPs at the MCSs, to
regulate lipid transport or directly move lipids between the apposed membranes of the
two organelles (Du, et al. 2011, Ngo & Ridgway. 2009).
35
2.5.5 ORPs act as sterol transporters The yeast ORP homologue Osh4p is able to transfer sterols from liposomes to
acceptor membranes. Moreover, Raychaudhuri et al. (2006) reported that depletion of
all seven Osh proteins decreased transfer of cholesterol or ergosterol from the PM to
the ER by 80%. Subsequently, Georgiev et al. (2011) used the fluorescent sterol
dehydroergosterol (DHE) to monitor the lipid traffic from the PM to the lipid droplets.
They found that cells deficient of all 7 Osh proteins displayed only a 50% reduction
of DHE transfer rate and a minor effect on ergosterol movement from the ER to the
PM. The authors thus concluded that the small effect in transport rate reflects the
dispensable role of Osh proteins for intracellular sterol transport, bringing up the
possibility that Osh proteins might rather regulate undefined downstream events.
Interestingly, de Saint-Jean et al. (2011) recently demonstrated that Osh4p efficiently
exchanges DHE for phosphatidylinositol-4-phosphate (PI4P) in vitro and is thus able
to transport these two lipids between membranes along opposite routes. The authors
proposed a model: on one hand, Osh4p transfer sterols from ER to the trans-Golgi; on
the other hand, it transports PI4P in the backward direction. The sterol/PI4P exchange
activity of Osh4p promotes sterol enrichment of membranes of the late secretory
pathway and regulates distribution of PI4P. In addition, most of the residues
responsible for PI4P recognition are conserved in Osh/ORP proteins, indicating that
ORPs potentially act as sterol/phosphoinositol phosphate exchangers (de Saint-Jean,
et al. 2011).
Several groups have reported that ORPs, OSBP, ORP1S, ORP2, ORP9L, and the
ORP5 ORD are able to transfer cholesterol between membranes in vitro (Du, et al.
2011, Ngo & Ridgway. 2009, Jansen, et al. 2011). However, only limited evidence
shows that ORPs play roles as cholesterol transporters in live cells. Du et al. (2011)
found that knocking down ORP5, an ER membrane protein, resulted in cholesterol
accumulation in late endosomes and lysosomes. The authors afterward proposed a
model that ORP5, in collaboration with NPC1, might regulate cholesterol transport
from the endosomes/lysosomes to the ER. Jansen et al. recently reported that
overexpression of two short ORPs, ORP1S and ORP2, significantly enhanced
cholesterol transport from the PM to the ER. Silencing of ORP1S and ORP2
simultaneously attenuated cholesterol transport from the PM to the ER and lipid
36
droplets (LDs), suggesting a function of these proteins in intracellular cholesterol
trafficking (Jansen, et al. 2011).
Despite the fact that purified ORPs can act as sterol transporters in vitro, scientists
have not reach a consensus on the putative role of ORPs as sterol transporters in vivo.
Schulz and colleagues found that all 7 Osh proteins could contact two distinct
membranes simultaneously, an activity which might efficiently regulate sterol
extraction and delivery between the donor and acceptor membranes (Schulz, et al.
2009). Through interaction with two membranes simultaneously, ORPs, hence, have
the potential to transport sterols at MCSs via flipping the sterols across the aqueous
phase. However, Geogiev et al. recently provided data suggesting that ORPs impact
sterol fluxes through altering the lateral organization of membrane lipids (Georgiev,
et al. 2011). Concerning the discrepant evidence, one cannot exclude the possibility
that ORPs could have an indirect, regulatory function in sterol trafficking.
2.5.6 ORPs regulate cellular lipid homeostasis OSBP overexpression was reported to stimulate cholesterol biosynthesis and reduce
cholesterol esterification (Lagace, et al. 1997). Cellular sterol status affects the
localization of OSBP at Golgi, suggesting that OSBP is a Golgi sterol content sensor.
In addition, OSBP is involved in activation of ceramide transport from ER to trans-
Golgi by CERT and SM synthesis (Perry & Ridgway. 2006, Banerji, et al. 2010).
OSBP phosphorylation by protein kinase D inhibits its Golgi localization in response
to 25-hydroxycholesterol stimulation or cholesterol depletion, impairs transport of
ceramide from the ER to the Golgi and induces Golgi fragmentation (Nhek, et al.
2010). Independently of this function, Bowden and Ridgway reported that OSBP
regulates cholesterol efflux and the stability of ATP-binding cassette transporter A1
(ABCA1) (Bowden & Ridgway. 2008). Furthermore, our group showed that
adenoviral overexpression of OSBP in mouse liver enhances plasma lipogenesis and
very-low-density lipoprotein (VLDL) secretion, the underlying mechanism apparently
being impact on the transcription factor SREBP-1c (Yan, et al. 2007b).
37
In addition to OSBP, increasing lines of evidence suggest that also other ORPs are
associated with lipid homeostasis. The localization of ORP2 on the surface of
cytoplasmic LDs depends on oxysterol binding (Hynynen, et al. 2009). Silencing of
ORP2 affects TG hydrolysis and cholesterol esterification, while overexpression of
ORP2 stimulates cellular cholesterol efflux (Hynynen, et al. 2005). Furthermore, our
group found that macrophage ORP1L overexpression in LDL receptor deficient mice
reduced cholesterol efflux to HDL and increased the size of atherosclerotic lesions
(Yan, et al. 2007a). Silencing of ORP1L in macrophage foam cells inhibits cholesterol
efflux to apolipoprotein A-I (apoA-I) (Vihervaara, et al. 2011b). Moreover, ORP8 in
human macrophages was reported to modulate ABCA1 expression and cholesterol
efflux to apoA-I (Yan, et al. 2008). Zhou et al. recently reported that ORP8
overexpression in mouse liver reduced levels of cholesterol, phospholipids and TG in
plasma and liver tissues. They found that ORP8 could moderately mediate the
expression of SREBP-1 and SREBP-2 (Zhou, et al. 2011). Interestingly, our group
recently demonstrated that silencing of 3 ORPs individually has distinct effects on the
lipidome of mouse macrophage cells (Vihervaara, et al. 2012). These findings
indicate that ORPs are involved in the control of cellular sterol and lipid homeostasis.
However, the underlying mechanisms remain poorly understood. It is likely that
altered sterol status or distribution upon ORP manipulation diversely affect a wide
range of events involved in lipid metabolism such as the endogenous oxysterol
syntheses, the lipid composition of subcellular membranes and the abundance of
MCSs.
2.5.7 The role of ORPs at MCSs (membrane contact sites) The ORP proteins comprise several motifs that control subcellular localization and
regulate their interactions with proteins or lipids (Figure 3). Most ORPs have an ER-
anchored fragment, either a FFAT motif which associates with ER membrane proteins,
VAPs or an ER-targeting membrane segment (Du, et al. 2011, Yan, et al. 2008,
Olkkonen, et al. 2012b). ER membrane, hence, is likely to be one target for the ORPs,
while PH domain in the N-terminal, in several cases, acts as a determinant for
targeting ORP proteins to a second membrane enriched in PIPs. This implies that
ORPs might, via membrane interactions, localize at MCSs between ER and the
38
apposed organelle (Figure 4). Schulz et al. reported that 4 Osh proteins, Osh2p, Osh3p,
Osh6p, and Osh7p, are enriched in ER-PM junctions (Schulz, et al. 2009). Stefan and
co-workers showed that the yeast ORP Osh3p acts at ER/PM contact sites as a sensor
of PM PI4P and an activator of the ER Sac1 phosphatase (Stefan, et al. 2011).
Moreover, Osh1p was found to localize at the nucleus-vacuole junction (NVJ), where
the nucleus and the vacuole are closely apposed. NVJ is a structure unique for yeast
cells, the formation of which, however, does not depend on Osh1p (Kvam & Goldfarb.
2004).
In mammalian cells, several lines of evidence suggest that some ORPs such as OSBP
(Peretti, et al. 2008), ORP1L (Rocha, et al. 2009, Vihervaara, et al. 2011b) and ORP5
(Du, et al. 2011) locate at MCSs. Three LTPs, OSBP, CERT and Nir2 at the ER–
Golgi membrane contact sites, coordinately regulate lipid composition of the Golgi
(Peretti, et al. 2008). Rocha and colleagues reported that ORP1L acts as a cholesterol
sensor that regulates the formation of ER-LE contact sites (Rocha, et al. 2009). Under
high cholesterol conditions, the Rab7 effector Rab7-interacting lysosomal protein
(RILP), in complex with ORP1L and Rab7, interacts with the subunit of dynein-
dynactin motor, p150(Glued). Under low cholesterol conditions, ORP1L undergoes a
conformational change to bind the ER membrane protein VAP, which induces the
formation of a junction between the ER and the LE, and removes p150(Glued) and
associated motors. ORP5 has a C-terminal trans-membrane segment which anchors it
in the ER. Moreover, ORP5 associates with the LE through an interaction with the
late endosomal cholesterol egress mediator NPC1 protein. Du et al. proposed a model
that ORP5 and NPC1 might coordinately induce the formation of LE-ER membrane
contact sites which facilitate the transport of endosomal cholesterol by ORP5 from LE
to the ER (Du, et al. 2011). These pieces of evidence suggest that certain ORP family
members are involved in MCS formation, or operate as regulatory factors or lipid
transporters at such sites.
2.5.8 ORPs modulate signaling processes OSBP acts as a cholesterol-sensing protein that regulates the activity of the
extracellular signal-activated kinases (ERK) signaling pathway (Wang, et al. 2005).
39
Furthermore, OSBP modifies JAK2/STAT3 signaling in response to 7-ketocholesterol,
which in turn controls the expression of the proatherogenic protein profilin-1 (Romeo
& Kazlauskas. 2008). Interestingly, increasing lines of evidence indicate that also
other members of the ORP family may exert similar scaffolding functions in cell
signaling pathways. Lessmann and coworkers first reported that ORP9 plays a role as
a novel modifier of the phosphatidylinositol 3-kinase (PI3K) pathway in different cell
types. According to them, ORP9 acts as a substrate of phosphoinositide-dependent
kinase-2 (PDK-2) to negatively regulate Akt phosphorylation (Lessmann, et al. 2007).
Furthermore, Goldfinger et al. identified ORP3 and ORP7 as R-Ras-interacting
proteins by immunoprecipitation and mass spectrometry (Goldfinger, et al. 2007). R-
Ras is a small GTPase of the Ras family that regulates cell adhesion, spreading and
migration. Interestingly, our group found that ORP3 exerts similar cellular effects as
R-Ras by regulating cell adhesion, spreading and integrin activity (Lehto, et al. 2008).
In addition, recent findings on yeast Osh proteins suggest that ORPs are potentially
involved in diverse signaling processes via modulation of PIP metabolism (de Saint-
Jean, et al. 2011).
Cholesterol, a major constituent of cell membrane, exerts essential functions in a wide
variety of signal transduction processes (Lingwood and Simons, 2010).
Overexpression or silencing of ORPs affects cholesterol distribution or intracellular
transportation (Hynynen, et al. 2009, Jansen, et al. 2011). Disturbance of ORP
functions, hence, is likely to impinge on signal transduction via modification of lipid
microdomain-associated processes. However, further investigations are required to
elucidate this issue in detail.
2.5.9 The roles of ORPs in human diseases The functions of ORPs proposed above raise the question of their putative roles in
disease. In fact, polymorphisms of several ORPs have already been reported to be
associated with metabolic disorders. Our group found that intronic variants of ORP10
associate with high plasma TG levels in Finnish dyslipidemic patients, the underlying
mechanism possibly involving a functional role of ORP10 in hepatocyte
apolipoprotein B-100 (apoB-100) secretion (Nissilä, et al. 2012, Perttilä, et al. 2009).
40
Subsequently, ORP10 variants were reported to associate with elevated plasma levels
of LDL-cholesterol (Koriyama, et al. 2010a) and peripheral artery disease (Koriyama,
et al. 2010b) in the Japanese population. These findings support an idea that ORP10
may play an important role in the development of dyslipidemias and atherosclerosis.
The closest relative of ORP10, ORP11, is analogously associated with risk factors of
metabolic diseases. Bouchard et al. reported that ORP11 was up-regulated in the
visceral adipose tissue of obese Canadian men with metabolic syndrome as compared
to obese subjects without the syndrome (Bouchard, et al. 2009). The authors further
showed that ORP11 variants in obese patients associated with a number of
cardiovascular risk factors such as LDL-cholesterol levels, diastolic blood pressure,
hyperglycemia/diabetes as well as metabolic syndrome per se. Subsequently, the
follow-up study by the same group showed that that ORP11 displayed different
expression levels in subcutaneous adipose tissue between the high and low responders
to caloric restriction (Bouchard, et al. 2010). The findings raise the possibility that
ORP11 is likely to be involved in the events occurring during adipocyte
differentiation which are connected with cardiovascular risk factors. Furthermore,
there is another ORP member, ORP7, the polymorphisms of which were reported to
be associated with serum total and LDL-cholesterol by genome-wide association
analysis (Teslovich, et al. 2010). However, there is no study assessing the functional
role of ORPs in adipocytes (Figure 4).
Alterations in the expression level of certain ORPs have been detected in cancerous
tumors or cells. For instance, OPR3 mRNA was found to be up-regulated in B-cell-
associate malignancies (Sander, et al. 2005, Chng, et al. 2006). Increased expression
of ORP4 mRNA was detected in circulating tumor cells in blood samples, indicating
that it might be a putative marker for tumor dissemination (Fournier, et al. 1999).
Koga et al. reported the association of ORP5 expression level with invasion and poor
prognosis of human pancreatic cancer (Koga, et al. 2008). These pieces of evidence
provide a notion that abnormal ORP expression levels may be functionally associated
with uncontrolled cell proliferation or tumor cell invasion.
41
Figure 4.
A schematic presentation summarizing the biological functions of ORPs. The
localization of ORPs at a membrane contact site is depicted. The role of ORPs in
adipose tissue is an unknown territory. ER, endoplasmic reticulum; VAP, VAMP-
associated protein anchoring the ORP at the ER; ORD, OSBP-related (ligand binding)
domain; PHD, pleckstrin homology domain; PIP, phosphatidylinositol phosphate.
2.6 GATE-16, GS28 and intracellular trafficking Several cellular proteins such as GATE-16 and GS28 are involved in the intracellular
trafficking. The Golgi SNARE complex 1 (GS28), which is also designated GOS28 or
GOSR1, is a 28-kDa Golgi vesicle SNARE (v-SNARE) protein. GS28 is involved in
intra-Golgi and ER–Golgi vesicular transport (Subramaniam, et al. 1996, Nagahama,
et al. 1996). GS28 is able to bind its cognate target SNARE (t-SNARE) syntaxin-5 at
the cis-Golgi and forms a SNARE complex for docking between donor and acceptor
membranes.
GATE-16 is a Golgi-associated ATPase enhancer that belongs to Autophagy-related 8
(Atg8) protein family. Mammalian Atg8 proteins comprise three subfamilies (Table
1), microtubule-associated protein 1 light chain 3 (LC3), γ-aminobutyric acid
receptor-associated protein (GABARAP) and GATE-16 (also called GABARAPL2)
(Shpilka, et al. 2011). GATE-16 is primarily expressed in the brain, similar to
42
GABARAP-L1, which is mainly expressed in the central nervous system. As an
essential factor for intra-Golgi protein transport, GATE-16 modulates late stages of
intra-Golgi protein transport by coupling N-ethylmaleimide sensitive factor (NSF)
activity and SNARE activation. Subsequently, soluble NSF attachment proteins
(SNAPs) bound to NSF, which facilitates the release of GATE-16 from the complex
and stimulate interaction of GATE-16 with GS28 in an ATP-dependent manner. The
specific interaction in turn precludes GS28 from binding its t-SNARE partner and
protects it from proteolysis (Sagiv, et al. 2000, Muller, et al. 2002). Therefore, GATE-
16 is likely to work as a v-SNARE protector and controls the appropriate assembly of
SNARE complexes.
Table 1. Human Autophagy-related 8 (Atg8) protein family
Subfamily Gene names Chromosome location
GATE-16 GATE-16/GABARAPL2/GEF2 16q22.1
GABARAP GABARAPL1/GEC1 12p13.31
GABARAP 17p13.1
LC3 MAP1LC3A 20q11.22
MAP1LC3B 16q24.2
MAP1LC3B2 12q24.22
MAP1LC3C 1q43
2.7 Adipocytes and the Simpson-Golabi-Behmel syndrome (SGBS) model The adipose tissue is the largest endocrine organ in human body, which comprises
20% of total body weight in lean adults and almost 50% or more in severely obese
individuals. The adipose tissue is constituted of multiple depots of white adipose
tissue and small depots of brown adipose tissue. White adipose tissue, a key endocrine
organ which contributes to energy storage and hormone production, is distributed in
43
the two major divisions throughout the body: subcutaneous adipose tissue and visceral
adipose tissue (also called abdominal or central adipose tissue). These two major
adipose tissues display distinct metabolic effects. Epidemiologists have demonstrated
that visceral adipose tissue exerts detrimental effects based on a positive association
between the tissue and metabolic disorders such as insulin resistance, T2D mellitus,
dyslipidemia, hypertension, atherosclerosis, and metabolic syndrome and further,
overall mortality (Wang, et al. 2005, Zhang, et al. 2008, Tran & Kahn. 2010). By
contrast, subcutaneous adipose tissue appears to be associated with insulin sensitivity
and a lower risk of T2D development, suggesting its protective role via improving
metabolic function (Misra, et al. 1997). Brown adipose tissue, which is innervated by
the sympathetic nervous system, is primarily responsible for energy expenditure and
heat production (Kozak & Young. 2012). As a highly vasculalized tissue, brown
adipose tissue is characterized by multilocular lipid droplets and abundant expression
of uncoupling protein-1 (UCP1) in the mitochondria (Gilsanz, et al. 2012).
A marked cellular heterogeneity is commonly found in adipose tissue: preadipocytes,
adipocytes, fibroblasts, macrophages, endothelial cells and multipotent stem cells are
the components of adipose tissue. Adipocytes, also called fat cells, are the major cells
in adipose tissue. In the medical field, increase in adipocyte number (hyperplasia) and
in adipocyte volume (hypertrophy) are two major types of processes to characterize
obesity. Hypertrophy is commonly detected in all overweight and obese individuals,
while hyperplasia is associated with severe obesity. At the cellular level, adipocytes
act as essential regulators of energy homeostasis. They secrete adipocyte-specific or
enriched protein factors that regulate a wide variety of processes such as energy
balance, metabolic events, immune function and angiogenesis. For instance, leptin is a
highly conserved hormone which is primarily expressed in adipose tissue and tightly
correlated with body mass index (BMI). Leptin acts as a regulator of food intake,
body weight, energy expenditure and normal immune function (Rajala & Scherer.
2003). Adiponectin, the most abundant hormone secreted from adipose tissue, is
negatively associated with adiposity, insulin resistance, T2D and metabolic syndrome
(Rabe, et al. 2008).
Adipocyte differentiation (adipogenesis) consists of two phases, determination and
termination. These processes involve complex gene-expression events which are
44
regulated by a cascade of transcription factors such as peroxisome proliferator-
activated receptor-γ (PPARγ). PPARγ, a member of the nuclear-receptor superfamily,
is a master regulator of adipocyte differentiation in vivo and in vitro. Apart from its
necessary role in adipogenesis, PPARγ is critical for maintenance of the differentiated
state of mature adipocytes (Rosen & MacDougald. 2006). Two kinds of cell lines,
preadipocyte and multipotent stem cell lines, are currently used to investigate the
molecular mechanisms involved in adipocyte differentiation. 3T3-L1 and 3T3-F442A,
mouse preadipocyte cell lines, are popular models to study the profile of adipocytes.
However, using rodent cells as models might produce different results compared with
ones derived from human source in respect to gene expression and regulation in
adipocytes. In 2001, Wabitsch’s group established the Simpson-Golabi-Behmel
syndrome (SGBS) pre-adipocyte model which could be effectively used to investigate
human adipocyte biology. SGBS cells are originated from subcutaneous adipose
tissue of an infant with Simson-Golabi-Behmel syndrome, an overgrowth syndrome
with macrostomia (large mouth), macroglossia (large tongue), hepatosplenomegaly
(enlarged liver and spleen), and renal and skeletal abnormalities (Wabitsch, et al.
2001, Fischer-Posovszky, et al. 2008). In the presence of PPARγ agonists in culture
medium without serum and albumin, the cells display efficient differentiation and
retain this capacity for up to 50 generations. The SGBS model, hence, has been
widely used to profile adipokine secretion, to identify drug effects on adipogenesis,
and to understand the mechanisms of human adipogenesis (Rosenow, et al. 2012,
Fischer-Posovszky, et al. 2012).
45
3 AIMS OF THE STUDY
I. To map the mRNA and protein expression patterns of OSBP/ORPs in
subcutaneous and visceral white fat of human subjects
II. To determine the functional effects of ORP genetic manipulation on the
adipocyte differentiation
III. To characterize the expression pattern, subcellular localization and function of
ORP11
IV. To identify protein binding partners of ORP11 and ORP7
V. To investigate the cellular role of ORP7
46
4 Materials and methods
4.1 List of Published methods
Method Original publication
Antibody generation
Western blot analysis
Immunohistochemistry*
Cell culture and transfection
Fluorescence microscopy
Electron microscopy*
RNA inference
Phospholipid binding assay
Yeast two-hybrid analysis*
Co-immunoprecipitation
Bimolecular fluorescence complementation (BIFC) assay*
Quantitative real-time PCR (qPCR) analysis
Human SGBS adipocytes culture
Lentiviral/adenoviral transduction
Oil Red O staining of neutral lipids
Cellular triglyceride assay
Bioinformatics analysis
Statistical analysis
I
I, II, III
I
I, II
I, II
I
I, II, III
I
I, II
I, II
II
II, III
III
III
III
III
II, III
III
*: methods used by collaborators
4.2 Brief descriptions of the methods employed Details regarding the materials and methods are presented in the original articles (I-
III).
47
Western blot analysis (I, II, III)
The cultured cells or tissues were homogenized in 250 mM Tris-HCl, pH 6.8, 8%
SDS and protease inhibitor cocktail. The crude extracts were cleared by centrifugation
and the protein concentration was subsequently measured. The proteins were
electrophoresed on Laemmli gels and electrotransferred onto Hybond-C Extra
nitrocellulose membranes. The bound primary antibodies were visualized with
horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) and the enhanced
chemiluminescence system. ImageJ (http://rsbweb.nih.gov/ij/) was used to quantify
the production levels after normalization of the data according to the β-actin signal.
Co-immunoprecipitation (I, II)
After transfection overnight, cells were washed twice with ice-cold PBS and
dissolved in the lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 0.5 mM MgCl2, 10%
glycerol, 0.5% Triton X-100, pH 8.0 and protease inhibitor cocktail). After
centrifugation, the supernatant was preabsorbed with protein G-Sepharose 4 Fast
Flow. The recovered supernatant was incubated with antibodies overnight and protein
G-Sepharose was added. Beads were washed three times with the lysis buffer and
boiled for western blot analysis.
Immunohistochemistry (II)
Immunohistochemical stainings were performed using affinity-purified rabbit
antibodies. The primary antibody was detected using the avidin-biotin complex
system. Brown 3,3’-diamino-benzidine was used as chromogen. After preincubation
with the immunizing polypeptide or with a non-related control polypeptide at 4 °C
overnight, the primary antibodies were used to simultaneously stain adjacent sections
in order to verify the specificity of the immunostaining signals.
48
Fluorescence microscopy (I, II)
For fixation, cells were fixed with 4% paraformaldehyde and then permeabilised with
0.1% Triton X-100 in PBS for 20 min. The cells were then incubated with primary
antibodies. After washing with PBS, the specimens were incubated with Alexa Fluor
secondary antibody conjugates. After washing with PBS, the specimens were
mounted on glass slides for the analysis by a laser scanning confocal microscope
system.
Electron microscopy (I)
The cells after different treatments were fixed in 2.5% glutaraldehyde and post-fixed
in 1% osmium tetroxide and 1.5% potassium ferrocyanide. Subsequently, the cell
specimens were dehydrated and embedded in the epoxy resin (Epon). Sections were
post-stained with uranyl acetate and lead citrate and analyzed with an electron
microscope.
Bimolecular fluorescence complementation (BiFC) assay (II)
Human BIFC constructs were generated to obtain fusions of the target genes with the
N- and C-terminal fragments of the fluorescent protein Venus. Plasmids were
transfected into the cells which were analyzed by a fluorescence microscope.
Yeast two-hybrid analysis (I and II)
Full-length human ORP11 and ORP7 cDNAs in the bait vector pGBKT7 were used to
screen a human normalized cDNA library (Clontech). Two-hybrid library screening
was conducted by using the yeast mating method. Briefly, the concentrated bait was
mixed with human cDNA library in yeast strains and incubated overnight until diploid
yeast clones formed. SD/-Trp-Leu-His-Ade (SD/4-) selective plates and β-
galactosidase (x-gal) filter assays were used to select positive colonies.
49
Quantitative RT-PCR (qPCR) analyses (II, III) Total RNAs from tissues or cells were reverse transcribed by using the VILO kit or
random hexanucleotide primers in the presence of RNase inhibitor (Takara Bio, Shiga,
Japan). The full-length ORP cDNA inserts were excised from constructs and used as
calibrators to estimate the corresponding mRNA copy numbers in tissues and cells as
previously described (Johansson, et al. 2003). Succinate dehydrogenase subunit alpha
(SDHA), β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were
used as housekeeping genes for the PCR runs. After measurement of threshold cycle
(CT) of each well, relative mRNA levels of target genes were quantified by using the
ΔΔCT method.
Human tissue specimens (I, III)
For immunohistochemistry, the specimens were obtained from medico-legal autopsies.
Human epicardial fat was obtained from a transplant recipient at Helsinki University
Central Hospital upon cardiac transplantation. Human subcutaneous and visceral
adipose tissue specimens were obtained from morbidly obese female patients upon
bariatric surgery at Peijas Hospital, Finland. The average BMI of the subjects (n=4)
was 46.9 kg/m2 (range 43.7 to 50.8 kg/m2) and waist to hip ratio 0.91 (range 0.88-
0.96).
Human SGBS preadipocyte/adipocyte culture (III)
Human SGBS preadipocytes were cultured as described by Wabitsch et al. (2001) and
Fischer-Posovszky et al. (2008). To induce differentiation, the cells at 80%-90%
confluence were incubated with serum-free DMEM/F12 medium supplemented with
0.01 mg/ml human apo-transferrin (Sigma-Aldrich), 20 nM insulin, 100 nM cortisol
(Sigma-Aldrich), 0.2 nM triiodothyronine (Sigma-Aldrich), 25 nM dexamethasone
(Sigma-Aldrich), 250 µM 1-methyl-3-isobutyl-xanthine (IBMX, Sigma-Aldrich) and
2 µM rosiglitazone (Sigma-Aldrich) for 4 days. Subsequently, the cells were changed
50
to serum-free DMEM/F12 medium containing 20 nM insulin, 100 nM cortisol, and
0.2 nM triiodothyronine and incubated for additional 18 days. In the adipocytes,
multiple lipid droplets were visible (Figure 5).
Figure 5.
Mature SGBS adipocytes after adipogenic differentiation. Oil Red O staining of
SGBS adipocytes differentiated for 22 days. The cells are filled with high amounts of
visible lipid droplets. A: scale bar = 400 µm. B: scale bar= 200 µm.
Lentiviral/adenoviral transduction (III)
SGBS cells were transduced with the shRNA lentiviruses for 72 hrs and stable cell
pools were selected by puromycine at 0.5 µg/ml. SGBS adipocytes under
differentiation on day 10 were transiently transduced by adenovirus at a multiplicity
of 50 PFU/cell for 72 hrs. The silencing or overexpression effects were verified by
qPCR and Western blotting.
Oil Red O staining of neutral lipids (III)
Cells were fixed with 10% formalin and subsequently washed with water. Then 60%
isopropanol was added on the cells for 5 min. 1.8 mg/ml Oil Red O in 60%
51
isopropanol was used to stain the cellular neutral lipids. Haematoxylin was used to
stain the nuclei of the cells.
Assay for cellular triglyceride content (III) The cells were lysed and the supernatant was harvested after centrifugation. A diluted
series of glycerol (2300, 1150, 575, 287.5, 143.75 and 71.88 µmol/L) was used as the
reference. Each sample was mixed with GPO-PAP Triglyceride kit (Cobas,
Roche/Hitachi) reagent on a 96-well plate for 20 min. The quantification of TG was
carried out by colorimetric assay at 510 nm. The triglyceride levels were normalized
according to total cell protein concentrations.
Bioinformatics analysis of OSBP/ORP mRNA expression (III) ORP mRNA expression was analysed based on the microarray dataset of Human
U133A/GNF1H Gene Atlas at the BioGPS web site (http://biogps.org/downloads/).
Information of the probe sets in different tissues was extracted separately according to
the individual ORP. In the case that the signal was throughout the tissue selection not
more than 3 folds from the median value, the probe set were estimated as “non-
unctional” and eliminated. The functionality of probe sets was further verified
according to the results on ORP mRNA or protein expression from previously
published reports. R program (http://www.r-project.org/) was used for the data
analysis and generation of a heat map.
Statistical analysis The data are presented as mean ± standard deviation (SD) in publication II and mean
± standard error of mean (SEM) in Publication III. Student’s T-test was applied for
comparisons between groups of data points. All statistical tests were two-sided and p
values <0.05 were considered significant.
52
5 RESULTS AND DISCUSSION
5.1 Characterization of ORP11
5.1.1 Tissue and cell type specific expression patterns of ORP11 ORP11 is a previously unexplored ORP family member. Using a 66-amino acid
region at the N-terminal of the protein, we generated an affinity-purified ORP11
rabbit antiserum for the characterization of ORP11. The Western blotting analysis of
the hepatoma cell line Huh7 and the embryonic kidney cell line HEK293 showed a
major protein band with the apparent molecular mass of 85 kDa. This is consistent
with the ORP11 mass predicted from the cDNA sequence, 83.6 kDa (I, Figure 2A).
On a commercial human multiple tissue blot filter, we detected ORP11 at highest
levels in ovary, testis, brain, kidney, liver and stomach, in contrast to its low levels
found in the small intestine, skeletal muscle, and spleen, and none in heart or lung (I,
Figure 1B). Brain is the most cholesterol-enriched organ in human. The oxysterol
24S-OHC is important for the elimination of the excess cholesterol in the brain
(Dietschy & Turley. 2004). Gonads are involved in extensive cholesterol uptake,
intracellular transport, and conversion to steroid hormones. Moreover, the central
roles of liver, kidney and adipose tissue in lipid homeostasis have been well accepted.
The abundant expression of ORP11 in these tissues would thus be consistent with a
role in active lipid metabolism or lipid sensing.
In addition, we observed abundant expression of ORP11 in a human pericardial
adipose tissue specimen by Western blotting (I, Figure 1B). Intriguingly, our
bioinformatics analysis showed that ORP11 is expressed in the adipocytes at
markedly high levels as compared to almost all the other tissues, based on analysis of
the human U1333A/GNF1H Gene Atlas microarray dataset (III, Figure S1). ORP11
was found abundantly expressed in visceral adipose tissue of obese Canadian men
with metabolic syndrome (Bouchard, et al. 2009). Therefore, we found it of interest to
investigate the functional role of ORP11 in adipose tissue.
53
The immunohistochemical staining of tissue sections from human autopsy material
displayed a strong ORP11 immunoreactivity in the epithelial cell layers of tubules in
kidney medulla (I, Figure 2A), of testicular seminiferous tubules (Figure 2B), of
caecum (I, Figure 2C), of skin epidermis and sebaceous gland (I, Figure 2D). The
abundant expression of the protein in epithelial cells indicates that ORP11 may play
pivotal roles in specific polarized cell types with the responsibility for directed
transport of cellular components. Skin epidermis and sebaceous gland epithelium are
important sites for lipid synthesis, storage or secretion, in which ORP11 might
regulate a wide variety of functions. Moreover, we observed strong immunostaining
in stroma of the ovary (I, Figure 2E), in liver hepatocytes (I, Figure 2F) as well as in
para-aortic lymph notes (Figure 6A) in contrast to the weak staining in sections of
skeletal muscle (Figure 6B) and lung (Figure 6C). Interestingly, the strongest staining
of ORP11 in liver hepatocytes localized to regions with lipid droplets reflecting
hepatic steatosis. Hence, excess neutral lipid accumulation might up-regulate or
stabilize ORP11. These findings suggest ORP11 may regulate cellular lipid
metabolism and are thus consistent with the previous findings that ORP11 gene
variants are associated with plasma LDL-cholesterol levels and several other
cardiovascular risk factors (Bouchard, et al. 2009).
Figure 6. Immunohistochemical analysis of ORP11 protein in human tissues
showing strong staining in para-aortic lymph notes (A) and weak staining in
skeletal muscle (B) and lung (C). Bar = 100 µm
54
5.1.2 Subcellular localization of ORP11
In contrast to most ORP family members, ORP11 lacks a FFAT motif which
potentially targets the ER. Immunofluorescence microscopy with the affinity-purified
ORP11 antiserum revealed that ORP11 localizes on Golgi membranes, specifically to
the medial-trans parts of the organelle (I, Figure 3). Moreover, we found that ORP11
showed significant co-localization with Rab6, Rab7 and Rab9 proteins, and
occasional co-localization with lysosome/late endosome markers, suggesting that
ORP11 localizes in the LE-TGN region (I, Figure 4). Rab7 and Rab9 are involved in
the communication between the LE and the Golgi complex. As a regulator of
membrane traffic, Rab9 binds to its effector TIP47, which enhances the recycling of
cation-independent mannose-6-phosphate receptor from the LE to the TGN
(Hutagalung & Novick. 2011). Rab7 plays a central role in trafficking from LE to
lysosome but also in LE-TGN recycling (Seaman, et al. 2009). ORPs are implicated
to be involved in non-vesicle communication rather than vesicle transport, which
might affect the lipid composition of subcellular organelle membranes (Rocha, et al.
2009, Olkkonen & Levine. 2004, Perry & Ridgway. 2006, Peretti, et al. 2008). Our
localization data suggested that ORP11 might play a role in non-vesicular
communication between the LE and the medial-trans-Golgi. Even though the
physiologic function of such communication has not been established, intimate
contacts between LE and trans-Golgi have been described in electron microscopic
studies at the so-called GERL (Golgi-ER-late endosome/lysosome) structures
(Novikoff, et al. 1971, Oliver, et al. 1980) in which lipid transport might occur
(Levine & Loewen. 2006).
Overexpression of EGFP-ORP11 resulted in an even cytosolic-appearing pattern with
a weak Golgi staining aspect. The cytosolic pattern suggests that ORP11 anchors at
the membrane at saturable sites. Unlike for the endogenous protein, we observed
significant colocalization between overexpressed ORP11 and all Golgi markers: the
cis-Golgi marker GM130 (I, Figure 5A-C), TGN marker TGN46 (I, Figure 5D-F) and
the medial-trans-Golgi marker GT-CHERRY (Figure 7A-C). The overexpressed
ORP11 also overlapped with GFP-Rab9 and GFP-Rab7, similarly to the endogenous
ORP11 (I, Figure 5J-O). The N-terminal fragment ORP11 (aa 1-292) displayed a
prominent LE localization and the C-terminal fragment ORP11 (aa 273-747) appeared
55
totally cytosolic, indicating that the C-terminal ORD domain negatively regulates the
LE targeting of ORP11. However, the PH domain failed to target the LE. This raises
the question how ORP11 targets the LE, which remains a subject of future studies.
Figure 7. Confocal microscopy images of HEK293 cells transfected with EGFP-
ORP11 and GT-CHERRY showing colocaliation at the medial-trans-Golgi. Bar =
10 µm
5.2 Cellular roles of ORP11
5.2.1 Overexpression of ORP11 induces the formation of lamellar lipid bodies
We frequently observed brightly stained vacuolar-like structures of ectopically
expressed ORP11, which colocalized significantly with GFP-Rab7 (I, Figure S1 G-I)
and GFP-Rab9 (I, Figure S1 J-L). Furthermore, electron microscopy showed
structures containing multilamellar membrane accumulation. The multilamellar lipid
bodies were found to be associated with vacuolar structures and the Golgi complex (I,
Figure S1). This indicates a disturbance of lipid trafficking on the endosome-Golgi
axis. Similar multilamellar membranes are formed in the endo-lysosomal system
where excess lipids accumulate because of disturbances in lipid transport. For
example, several group observed multilamellar membranes arises upon incubation
with oxidized LDL or induced by inheritable lysosomal storage disorders (Anderson
& Borlak. 2006, Schmitz & Grandl. 2009b). Moreover, this finding is in good
agreement with the localization data of ORP11 in the LE-Golgi region.
56
The localization of OSBP and ORP9 to the TGN is mediated by their PH domains,
which bind to PI(4)P. An in vitro binding assay showed that a GST fusion protein of
the ORP11 the PH domain (aa 60-157) specifically bound to PtdIns(3)P, -(4)P, -(5)P,
-(3,4)P2, (4,5)P2, and phosphatidic acid (I, Figure 7A). A fluorescent fusion carrying
two ORP11 PH domains in tandem appeared largely cytosolic and intra-nuclear and
occasionally displayed weak staining in the Golgi (I, Figure 7B-D). Hence, the
interaction between the PH domain and PI(4)P might partially regulate ORP11
targeting to the Golgi, but cannot be entirely responsible for this localization.
5.2.2 ORP11 interacts with ORP9
To identify the determinants that might impact the cellular localization of ORP11, we
carried out a yeast two-hybrid analysis using full-length ORP11 as a bait to screen a
library consisting of human cDNAs from a wide spectrum of tissues. ORP9 was
identified as one candidate to interact with ORP11. Immunoprecipitation using lysate
of HEK293 cells after cotransfection with Xpress-ORP11 and Flag-tagged ORP9
further confirmed the interaction between the two proteins (I, Figure 8A).
To determine the specific interacting region, we made serial deletion constructs of
ORP9 in the prey plasmid pGADT7 and of ORP11 in the bait vector pGBKT7 (I, Fig
9A, C). The yeast two-hybrid assay showed that aa 154-747 of ORP11 dimerized with
ORP9 within aa 98-372, possibly 106-195 (I, Figure 9B, D). The cutpoint at aa 367
aligned exactly with the cutpoint 372 in ORP9. However, two-hybrid tests failed to
detect interaction between the N-terminal ORP11(1-367) and ORP9. Interestingly
however, the N-terminal fragment ORP11(1-292) could be co-immunoprecipitated
with ORP9 (I, Figure 9E), suggesting that the result in the two-hybrid assay with
ORP11(1-367) was a false negative. Taken together, ORP11 aa 154-29 is most likely
to be the dimerization region.
ORP9 has a FFAT motif and a PH domain which determine the targeting of the
protein to ER and Golgi membranes (Ngo & Ridgway. 2009, Wyles & Ridgway.
2004). Overexpression of ORP9 recruited the largely cytosolic EGFP-ORP11 to the
ER and Golgi membranes (I, Figure 8B-D). Almost 100% overlap between ORP9 and
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ORP11 demonstrated that the recruitment of ORP11 by ORP9 is highly efficient,
indicating that the presence of ORP9 protein in trans-Golgi may contribute to the
ORP11 Golgi association. ORP11 has no FFAT motif that would specifically interact
with ER membrane protein VAP-A. Our results confirmed that ORP11 has no
association with ER membranes via VAP-A, and suggest that ORP9 recruits ORP11
directly to specific membranes (I, Figure 8E-G).
5.2.3 ORP11 Golgi association is facilitated by ORP9
The expression of ORP9 protein was reduced by 85-90%, while ORP11 was reduced
by 75-80% in HEK293 cells after siRNA transfections (I, Figure 10A). After
significant reduction of ORP9, we failed to observe the typical distribution of ORP11
in the Golgi. Instead, we detected ORP11 distributed in dispersed, vesicular structures
(I, Figure 10D and E) different from the control (I, Figure 10C), which might be
caused by a shift towards an endosomal localization. However, remarkable reduction
of ORP11 had no effect on the distribution of ORP9 (I, Figure 10F and G), which
remained similar to the control (Figure 10B). Furthermore, Western blotting analysis
showed that silencing of ORP9 or ORP11 had no impact on the levels of the other
protein in the cells (I, Figure 10A).
Ridgway et al. observed Golgi fragmentation after knockdown of ORP9L expression
(Ngo & Ridgway. 2009). In our hands ORP9 deficiency had only a minor effect on
TGN integrity as judged from co-staining for TGN46 (Figure 8A-C), and absolutely
no effect on cis-Golgi morphology, as judged from GM130 staining (data not shown).
At the level of ORP11 silencing reached we observed no disturbance of Golgi
complex integrity by reduction of the cellular ORP11 protein (Figure 8D-F).
Overexpressed ORP9 was able to recruit ORP11 to ER and Golgi membranes through
dimerization between the two proteins, suggesting that the presence of ORP9 in trans-
Golgi might regulate ORP11 targeting. Silencing of ORP9 resulted in a dispersed,
vesicular distribution of ORP11, supporting the important impact of ORP9 on ORP11
Golgi association. Hence, Golgi localization of ORP11 appears to be determined by
complex membrane targeting through dimerization with ORP9 and interaction of the
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PH domain with acidic phospholipids. Interestingly, the localizations of certain other
ORPs are reported to be determined in a similar manner, by complex membrane
targeting. For instance, interactions with PI(4)P and with the small GTPase ARF drive
the localization of OSBP to TGN membranes (Levine et al., 1998; Levine et al., 2002).
The ankyrin repeat region of ORP11 interacts with Rab7 and the PH domain binds to
phosphoinositides, which contribute to the localization of ORP1L to the LE
(Johansson, et al. 2003, Johansson, et al. 2005).
Figure 8. Confocal microscopy images of HEK293 cells transfected with siORP9
and siORP11 respectively showing silencing ORP9 or ORP11 has no effect on
Golgi complex integrity. A-C. Treatment with siORP9, co-staining for ORP9 and
TGN46. D-E. Treatment with siORP11, double staining for ORP11 and TGN46. Bar
= 20 µm.
5.3 ORPs in human adipose depots and cultured adipocytes
OSBP acts as a sterol sensor that integrates the cellular sterol status with
sphingomyelin metabolism by regulating ceramide transport from the endoplasmic
59
reticulum (ER) to the Golgi apparatus (Perry & Ridgway. 2006). OSBP, ORP1L,
ORP2, and ORP8 play important roles in cellular cholesterol and triglyceride
metabolism (Yan, et al. 2007a, Hynynen, et al. 2009, Yan, et al. 2008, Yan, et al.
2007b, Hynynen, et al. 2005). Very recently, ORP10, a close relative of ORP11, was
reported to negatively regulate hepatocyte β-lipoprotein secretion, which might
contribute to the association of OSBPL10 genetic variation with dyslipidemias and
atherosclerosis (Nissilä, et al. 2012, Perttilä, et al. 2009). Increasing lines of evidence
suggest that members of the ORP family sense the intracellular concentrations of
different sterols and relay information to machineries responsible for the control of
cellular lipid homeostasis, intracellular vesicle transport, and cell signaling.
5.3.1 OSBP/ORP copy numbers in human adipose depots and SGBS adipocytes. To date, no group has compared the expression of OSBP/ORP in human visceral and
subcutaneous adipose tissues, or assessed the functional role of ORPs in adipocytes.
We measured copy numbers of ORP mRNAs in human subcutaneous and visceral
adipose tissue specimens from 4 obese female patients, as well as in Simpson-Golabi-
Behmel syndrome (SGBS) cells. We found that OSBP/ORP displayed almost
identical expression patterns in the subcutaneous (s.c.) and visceral fat depots (III,
Figure 1A, B), and a highly similar pattern in the SGBS adipocytes (III, Figure 1C).
This suggests that the ORP mRNA signals in the human adipose tissue specimens
mainly derive from adipocytes, and are not strongly affected by mRNAs of ORPs
from other cell types included in the adipose tissues such as fibroblasts, macrophages,
and endothelial cells (Gimeno & Klaman. 2005). In fact, the expression profiles of
different ORPs were markedly uneven in the adipose tissues, with up to 100-fold
differences between the distinct ORP mRNAs detected.
Among the ORPs the mRNAs of which were abundant in the adipose depots (III,
Figure 1A, B), OSBP, ORP2, ORP8, and ORP9 were detected at relatively equal
levels in the two depots by Western blotting of total protein specimens (III, Figure 2).
Although ORP11 mRNA was present at relatively low levels in the adipose depots,
we were able to detect the protein by Western blotting indicating that our antiserum
was highly sensitive.
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Bioinformatics analysis of human U133A/GNF1H Gene Atlas dataset showed that
OSBP, ORP4 and ORP9 mRNAs were expressed at relatively even levels across
different tissues and cell types (III, Figure S1). ORP8 was found abundantly
expressed in adipocytes, CD14(+) monocytes (III, Figure S1) and in tissue
macrophage (result by Yan et al., 2008), suggesting that a proportion of the ORP8
mRNA signals in the adipose depots of patients might come from monocyte-
macrophages within the inflamed adipose tissues.
Interestingly, even though ORP11 has a relative low mRNA copy number in adipose
depots, its mRNA expression in adipocytes was markedly higher as compared to most
of the other tissues/cell types, suggesting that it might have important specific
functions in the adipocytes. Importantly, ORP11 mRNA was found to be elevated in
the visceral adipose tissue of obese patients with metabolic syndrome compared to the
obese individuals without the syndrome, bringing up the possibility that ORP11 might
adversely affect adipose tissue metabolism or signaling (Bouchard, et al. 2009).
5.3.2 OSBP/ORP expression during SGBS adipocyte differentiation After 22 days of differentiation, we observed that 80% of SGBS cells were filled with
lipid droplets (III, Figure 3A, B). In good accordance with previous reports (Wabitsch
et al., 2001; Fischer-Posovszky et al., 2008), extensive up-regulation of adiponectin,
aP2, leptin and PPARγ at the mRNA level was detected (III, Figure 3C), consistent
with the view that a large proportion of the cells were differentiated to adipocytes.
As compared to preadipocytes, five ORPs, ORP2 (-54%), ORP3 (-86%), ORP4 (-
74%), ORP7 (-56%), and ORP8 (-67%) were significantly down-regulated at the
mRNA level in adipocytes. In contrast, only one mRNA, ORP11, was upregulated
3.5-fold upon SGBS cell adipocytic differentiaton (III, Figure 4A). Of these six ORPs,
ORP3, ORP8 and ORP11 were selected as the candidates for the further analysis due
to the availability of excellent molecular and immunological tools in the lab.
Furthermore, we observed by Western blotting a 2-fold increase of ORP11 protein, a
55% reduction of ORP8, and a 60% reduction of ORP3 upon SGBS adipocytic
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differentiation (III, Figure 4B, C), confirming the changes in ORP3, ORP8 and
ORP11 expression at the protein level (Figure 4B).
Time course data showed that the down-regulation of ORP3 and ORP8 occurred
between days 0 and 4 without further progress during the late stages. This might be
associated with the cessation of cell proliferation due to the adipogenesis. However,
the ORP11 mRNA was elevated at the late time points between days 16 and 22. It is
thus likely that ORP11 exerts a role during late stages of adipogenesis, or might be
essential for maintenance of the mature adipocyte phenotype.
5.3.3 Effect of ORP silencing/overexpression on adipocyte differentiation
ORP11 was overexpressed in the visceral adipose tissue of obese Canadian men with
metabolic syndrome (Bouchard, et al. 2009). We found during this study ORP11
localizes at the Golgi-late endosome interface in HEK293 cells, and demonstrated its
dimerization with ORP9 (I). However, the precise function of ORP11 remains
obscure.
Transduction of SGBS preadipocytes with a lentivirus expressing ORP11 shRNA
resulted in 60-70% reduction of the ORP11 protein as compared to the controls (III,
Figure 6A). Interestingly, a significant reduction of adiponectin and aP2 mRNAs was
detected in the ORP11 knock-down cells as compared to the controls (III, Figure 6B).
Adiponectin is an adipocyte-specific protein, which in humans is encoded by the
ADIPOQ gene (Scherer, et al. 1995). As an abundant circulating adipocytokine,
adiponectin has anti-inflammatory properties negatively associated with
cardiovascular disease, T2D, and obesity. Because of its anti-inflammatory and anti-
atherogenic effects, adiponectin is commonly considered an important potential target
for pharmacologic treatment of T2D and metabolic syndrome (Lago, et al. 2007). aP2,
also called FABP4, is a 15-kD cytosolic fatty acid binding protein which is primarily
expressed in adipocytes and macrophages (Coe, et al. 1999). The protein has
important regulatory functions in glucose and lipid homeostasis. Lipolysis efficiency
was reported to be markedly reduced in the adipocytes derived from aP2-deficient
mice (Coe, et al. 1999). Increasing lines of evidence suggest that aP2 is closely
62
associated with insulin resistance, metabolic syndrome, type 2 diabetes, and
atherosclerosis (Terra, et al. 2011). In addition, serum aP2 level is also associated
with the development of metabolic syndrome, T2D and atherogenic dyslipidemia (Xu,
et al. 2007, Tso, et al. 2007, Cabre, et al. 2012). Apart from the impacts on
adiponectin and aP2 expression, silencing of ORP11 markedly reduced the storage of
triglycerides in the SGBS adipocytes, as compared to the controls transduced with a
non-targeting shRNA lentivirus (III, Fig 6C). In addition, a HuH7 hepatoma cell line
with stably silenced ORP11 was generated by lentiviral transduction. ORP11 was
stably silenced at least until passage 10 (Figure 9). This stable hepatoma cell model
could be used to investigate potential role of ORP11 in hepatocytes.
Figure 9. The Western blotting analysis of Huh7 cells demonstrates that ORP11
was stably silenced until passage 10. HuH7 cells were transduced with an ORP11
shRNA (Sh-ORP11) or non-targeting (Sh-NT) shRNA lentivirus. P3, P6, P8, P10:
passage 3, passage 6, passage 8 and passage 10.
According to the results of the differentiation time course experiments (III, Figure 5),
endogeneous ORP3 and ORP 8 mRNAs were firmly down-regulated and cytoplasmic
LDs were rapidly generated on day 10 (III, Figure 5). Therefore, we started to
overexpress ORP3 or ORP8 on this time point of SGSB adipogenesis. After 3 days'
transduction, ORP8 protein was markedly overexpressed (III, Figure 6A), resulted in
a significant reduction of ap2 mRNA levels, and impaired the storage of cellular
triglycerides (III, Figure 6B). ORP8 has been reported to regulate lipid metabolism
63
and nuclear functions (Beaslas, et al. 2012). As a target of microRNA (miR)-143,
ORP8 exerts an important function to mediate insulin signaling in mouse adipocytes
(Jordan, et al. 2011). Therefore, ORP8 overexpression might impair the insulin
stimulation of the adipogenic process. Further studies on the connection between
ORP8 and insulin signaling pathways in adipocytes are therefore warranted.
In contrast, ORP3 overexpression affected significantly neither mRNA levels of
adipogenesis markers mRNA levels nor cellular triglyceride storage (III, Figure 6B),
suggesting a non-essential role of ORP3 upon the adipogenesis. As a phosphoprotein,
ORP3 regulates cell adhesion (Lehto, et al. 2008). ORP3 protein was reported to be
up-regulated in adipocytes differentiated from human mesenchymal stem cells, which
is inconsistent with our results (Lee, et al. 2006). The use of different cell models in
the two studies is mostly likely to result in the inconsistency. We found that ORP3
was abundantly expressed in the adipose depots and adipocytes, which is consistent
with the results in (Lee, et al. 2006). We therefore find it likely that ORP3 could be
up-regulated at an earlier phase of mesenchymal stem differentiation into the
adipocytic lineage, and not at the preadipocyte-adipocyte conversion.
5.4 ORP7 and GATE-16
5.4.1 ORP7 interacts with GATE-16 ORP7 was found predominantly expressed in gastrointestinal tract including stomach,
duodenum, jejunum and ascending colon as well as in fetal lung. In human intestinal
samples, ORP7 is exclusively expressed in the epithelial cells indicating that it might
be involved in the lipid absorption occurring at the intestinal epithelium (Lehto, et al.
2004). It was previously reported that ORP7 protein is distributed between the cytosol
and ER membranes (Lehto, et al. 2004).
Using full-length ORP7 as bait and a normalized universal human cDNA library as
prey, we carried out a yeast two-hybrid screen. We identified two positive clones
encoding GATE-16, which is a Golgi-associated ATPase enhancer (Sagiv, et al.
2000). The results from yeast two-hybrid analysis provided a clue that ORP7 interacts
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with GATE-16. BiFC assay further confirmed the interaction in live cells, which is
visualized in the figure (II, Figure 1C, D).
To identify specific regions in GATE-16 and ORP7 responsible for the interaction, we
made serial deletion constructs of GATE-16 in the prey vector pGADT7 (II, Figure
3A) and of ORP7 in the bait plasmid pGBKT7 (II, Figure 3C). In combination with
empty prey vector, neither full length nor deletion constructs of ORP7 bait could
grow on SD/4- agar plates or activate the β-gal reporter gene. As prey constructs,
GATE-16 (aa 30-117) but not GATE-16 (1-45) were able to grow on SD/4- agar
plates and to activate the transcription of β-gal, suggesting that amino acids 30-117 of
GATE-16 interact with ORP7 (II, Figure 3B). As the bait, ORP7 (aa 1-142) and
ORP7 (aa 1-472) constructs grew on SD/4- agar and activated transcription of the β-
gal reporter gene. In contrast, amino acids 470-842 and 142-828 in the C-terminal
region containing the ORD of ORP7 failed to activate the reporter (II, Figure 3D).
This finding implicates that amino acids 1-142 of ORP7 are sufficient to mediate its
interaction with GATE-16. The binding region of ORP7 contains its PH domain
which might play a role as a determinant for membrane targeting via interaction with
phospholipids and proteins such as GATE-16 (Vihervaara, et al. 2011a).
5.4.2 Subcellular localization of ORP7, GATE-16 and GS28 As a member of the ubiquitin-fold (UF) protein family, GATE-16 is located to the
Golgi complex and interacts with GS28 (Muller, et al. 2002). In 293A cells,
overexpressed GFP-GATE-16 displayed a pattern consisting of largely cytosolic
distribution with some Golgi staining (II, Figure 2A-C). Overexpressed ORP7
displayed some ER-like staining but was prominently distributed as large, vacuolar-
like elements co-localizing with the autophagosome marker RFP-LC3 (II, Figure 2D-
F). However, overexpression of RFP-LC3 alone resulted in a pattern consisting of
largely cytosolic staining and small punctate structures, smaller than the ones in the
presence of the excess ORP7 (II, Figure 2G-I). The findings implicated that
overexpressed ORP7 induced formation of vacuolar-like structures representing
autophagosomes.
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As described in 5.4.1, ORP7 interacts with GATE-16. We further investigated the
impact of the interaction on their localization. The brightly stained vacuolar-like
structures represented ORP7 co-localizing strongly with GFP-GATE-16 (II, Figure
2J-L). In contrast, the endogeneous GS28 did not overlap with vacuolar-like elements
containing overexpressed ORP7 (II, Figure 2M-O). This suggests that ORP7 is able to
recruit GATE-16 but not GS28 to the large autophagosomal structures.
5.4.3 ORP7 regulates GS28 protein level through transcriptional regulation ORP7 overexpression reduced the GS28 protein level by 25% in 293A cells (II,
Figure 4C, E). Moreover, the ORP7 protein was reduced by approximately 90% after
transfection of the most efficient ORP7-specific shRNA (sh3638) (II, Figure 4A),
which resulted in a 40% upregulation of cellular GS28 protein (II, Figure 4B, D).
Therefore, not only the excess ORP7 upon overexpression but also the endogenous
ORP7 modulates the stability of GS28 protein. However, overexpression of ORP7
failed to reduce the mRNA levels of GS28 as compared to the control (II, Figure 5F),
suggesting that the regulation operates through a post-transcriptional mechanism.
5.4.4 ORP7 affects GS28 degradation on proteasomes via GATE-16
As a component of the intra-Golgi transport machinery, GATE-16 binds to GS28, a
Golgi v-SNARE in an ATP-dependent but not ATP hydrolysis dependent manner,
protecting GS28 from proteolysis and maintaining GS28 in a competent state for
promoting Golgi vesicle transport (Sagiv, et al. 2000, Muller, et al. 2002). We found
that silencing of GATE-16 resulted in a significant reduction of GS28 by 85% (II,
Figure 5A, C, E).
To investigate whether GATE-16 regulates the effect of ORP7 on GS28 protein levels,
we generated a truncated construct of ORP7 (aa 1-142) lacking the region binding to
GATE-16. Interestingly, overexpression of the truncated construct had no effect on
GS28 protein levels, differing from the impact of full-length ORP7 protein (II, Figure
5B, D). The finding suggests that GATE-16 is involved in the functional crosstalk
between ORP7 and GS28.
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Based on the previous results, we envisioned that ORP7 and GS28 might act as
competitive partners for GATE-16. Their competitive interactions with GATE-16
might attenuate the protection of GATE-16 on GS28 and in turn result in the
proteolysis of GS28. To investigate whether the reduction of GS28 follows the
mechanism of proteasome-dependent degradation or lysosomal degradation, we
treated cells with the proteasome inhibitor MG132 or the lysosomal inhibitor
chloroquine, respectively. MG132 effectively blocks proteolytic activity of the
proteasome complex (Myung, et al. 2001), while chloroquine accumulates in
lysosomes and impairs lysosomal protein degradation via inhibition of endo-
lysosomal acidification (Gonzalez-Noriega, et al. 1980). Overexpression of ORP7
resulted in a significant reduction of GS28 protein levels, consistent with the result in
5.4.3. MG132 treatment for 16 h significantly inhibited the degradation of GS28 (III,
Figure 5G). In contrast, in the cells treated with chloroquine, GS28 was degraded as
efficiently as in the controls. The results suggest that ORP7 affects GS28 protein
levels via a mechanism of proteasomal and not lysosomal degradation.
5.4.5 ORP7 potentiates 25-OH-induced degradation of GS28 After treatment of cells with serial dilutions of 25-OHC for 48 h, we observed a
inverse association between GS28 protein levels and 25-OHC concentrations, as
compared to the control (II, Figure 6A, C). Increasing 25-OHC concentrations
induced reduction of GS28 protein, an effect that was further strengthened upon
overexpression of ORP7 (II, Figure 6B, C). In contrast, silencing of ORP7 abolished
the impact of 25-OHC on GS28 protein levels (II, Figure 6D-F). Thus ORP7
overexpressing and silencing strengthen or abolish the effect of 25-OHC,
demonstrating that ORP7 acts a potential mediator of the oxysterol effect of GS28
stability.
To investigate whether GS28 degradation upon 25-OHC treatment is due to
proteasomal or lysosomal degradation, we treated the cells with MG132 or
chloroquine similarly to the previous experiment. GS28 protein degradation was
eliminated after treatment with MG132 but not chloroquine, indicating that 25-OHC
induces GS28 degradation which is dependent on the proteasome but not lysosome
hydrolytic activity.
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When 25-OHC or cholesterol binds to the ORD domain of OSBP, the protein
undergoes a conformational change to facilitate its cellular function (Wang, et al.
2005, Wang, et al. 2008). ORP7 has been reported to have the capacity to bind to 25-
OHC (Suchanek, et al. 2007). Interestingly, 25-OHC was reported to diminish the
GS28 protein but not the lysosome-associated protein LAMP1 in melanocytes
through an unknown mechanism (Hall, et al. 2004). We postulated, therefore, that 25-
OHC might regulate the function of ORP7 in a similar manner to OSBP. ORP7 could
undergo a conformational change upon binding of 25-OHC in the ORD region. The
conformational change may enhance the affinity of ORP7 binding to GATE-16,
which competitively abolishes the protection of GS28 afforded by GATE-16 and
results in the proteolysis of the t-SNARE. Therefore, the crosstalk between ORP7 and
its oxysterol ligand can be envisioned to maintain the stability of GS28.
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6 CONCLUSIONS AND PERSPECTIVES
Metabolic disorders such as obesity, metabolic syndrome, T2D, dyslipidemia,
hypertension and atherosclerosis seriously erode human life quality. They result in an
extremely heavy burden on global socioeconomics because their characteristics - high
prevalence and incidence, chronicity, and long-term health care implications. Obesity
is an essential independent risk factor of cardiovascular disease, T2D and certain
other chronic disorders. It is characterized by status, distribution and amount of
adipose tissue which is the largest endocrine organ in the human body. The function
of this endocrine organ is primarily regulated by a variety of events occurring in the
adipocytes. According to the previous evidences, OSBP/ORP proteins sense the
intracellular concentrations of different sterols and relay information to cellular
machineries that contribute to cellular lipid homeostasis, intracellular vesicle transport,
and cell signaling. Disturbances of these functions result in the interruption of cellular
events and are most likely to play critical roles in metabolic disorders.
Data presented in this thesis showed that OSBP/ORPs have similar mRNA expression
patterns in the human s.c. and visceral adipose depots, as well as in human SGBS
adipocytes. During adipocytes differentiation, ORP2, ORP3, ORP4, ORP7 and ORP8
mRNA levels are upregulated while ORP11 is downregulated. Importantly, this work
is the first one to reveal the function role of ORP proteins during adipocytes
differentiation: ORP8 affects aP2 expression and ORP11 modulates aP2 and
adiponection mRNA levels, and both of them play roles as regulators of adipocyte TG
storage. Moreover, disturbance of the functions of ORP proteins might have the
potential to influence expression of some other key genes and additional cellular
events during adipogenesis. Further study is hence warranted.
According to our data, OPR11, among all the ORP family members, is the only one
induced during adipogenesis. The present work reports the tissue and cell type-
specific expression patterns of ORP11. The protein localizes in the Golgi-LE interface
and dimerizes with its close relative ORP9. The dimerization regulates the subcellular
targeting of ORP11. Therefore, we envision that the ORP9-ORP11 dimer might act as
a sterol sensor or transporter to regulate lipid homeostasis and further influence
69
metabolic disorders. It will be interesting to investigate whether the dimerization of
ORP11 with ORP9 plays a role in adipocyte differentiation.
Common ORP7 genetic variants significantly associate with plasma LDL and total
cholesterol in large population samples, indicating that also this protein might be
functionally correlated with metabolic disorders (Teslovich, et al. 2010). Here, we
first report that GATE-16 as a binding partner of ORP7. The work in this thesis
reveals that ORP7 modulates the stability of GS28 via binding to GATE-16.
Moreover, ORP7 modulates the effect of its ligand 25-OHC on proteasome-dependent
degradation of GS28, which may result in modulation of Golgi transport functions.
As a conclusion, the lines of evidence from previous reports and this thesis suggest
that ORPs regulate lipid metabolism, intracellular membrane trafficking and
adipocyte differentiation via dimerization with their homologues, interactions with
specific membrane proteins and lipid components, and via regulatory impacts on
proteins characteristic of adipogenesis. Further exploration of the detailed
mechanisms of ORP action and their roles in molecular pathways disturbed in human
disease are warranted in future studies.
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7 ACKNOWLEDGEMENTS
“It is good to have an end to journey toward; but it is the journey that matters, in the
end.”
- Ernest Hemingway
The completion of this PhD thesis represents the landmark of a long journey’s end.
However, I believe the challenging journey is best measured in friends rather than
miles. It was only through the help and support from the following people that I could
walk along this arduous but fulfilling road and reach the end.
I would like to express my heartfelt gratitude to my supervisor, Adjunct Professor
Vesa Olkkonen for his thoughtful guidance, warm encouragement and constant
support. What I learned from him is not only scientific knowledge but also organizing
skills, sanguine attitude, great patience and scientific motivation. I deeply thank him
for encouraging me to take a variety of lectures and seminars and guild me to
organize multiple tasks effectively: You set up as one of the best role models in my
life.
I wish to express my deep gratitude to Adjunct Professor Anna-Liisa Levonen for
being as my opponent and Prof. Elina Ikonen as my Kustos. I gratefully
acknowledge Prof. Pekka Lappalainen and Adjunct Professor Pirkko Pussinen for
reviewing the thesis and for constructive discussions and valuable suggestions in such
a tight schedule. I warmly thank my thesis committee members, Adjunct Professor
Matti Jauhiainen and Dr. Saara Laitinen for your great contribution on following
up the progress of my work.
Without excellent technical support from Seikku Puomilahti, Liisa Arala, Eeva
Jääskeläinen, Pirjo Ranta, Sari Nuutinen and Jari Metso, close collaboration with
Prof. Daguang Yan, Prof. Hannele Yki-Järvinen, Prof. Christian Ehnholm, Prof.
Martin Wabitsch, Marius Robciuc, Shiqian Li, Mikko Mäyränpää, Nils Bäck,
Wenbin Zhong, and effective secretarial assistances from Carita and Cia, no piece
71
of work in this thesis could be completed. I owe a great deal of appreciation for all of
you.
Words are short to express my deep sense of gratitude towards my present and former
colleagues, Marion, Markku, Olivier, Terhi, Julia, Eija, Riikka, Raghu,
Henriikka, Hanna, Raisa and all the members in Minerva. I am grateful for Jarkko
and Pirkka-Pekka for creating funny topics. I would like to express my sincere
thanks to my present and former “office mates”, Jenny, Noora, Alise, Sami, Jani and
Timofey for all the pleasing time.
This work was carried out in the National Institute for Health and Welfare (2007-2009)
and Minerva Foundation Institute for Medical Research (2010-2012). I would like to
thank following organisations for their generous financial support of the work: the
Sigrid Jusélius Foundation, the Finnish Foundation for Cardiovascular Research, the
Magnus Ehrnrooth Foundation, the Liv och Hälsa Foundation, the Novo Nordisk
Foundation, the Academy of Finland, the EU FP7 (LipidomicNet), the Paulo
Foundation, the Orion-Farmos Research Foundation, the University of Helsinki
Medicine Fund and Jubilee Fund, the Finnish Atherosclerosis Society and the
University of Helsinki Chancellor travel grant.
I am cordially thankful to Prof. Deyin Guo of Wuhan University, for his warm advice
and support during my master study from 2005-2007. Dr. Chunmei Li is deeply
thanked for her kind help in Finland. My thanks go to all my Chinese and Finnish
friends, in particular to Lu Cheng & Danmei Huang, Erkka & Anna-Maria
Tuomela.
I would like to extend huge, warm thanks to all the Finnish people – this friendly and
respectful nation plays one of the most influential part in my life. Our baby, hence,
has a Finnish name, Joel.
The road of the PhD journey would not be contemplated without thoughtful love and
encouragement from my parents, Jian-ping Zhou and Li Wang as well as my
parents-in-law, Wan Goo Cho and Kyung Hee Cho. To all of you, a thousand
thanks!
72
At last but not least, to Eunjee for sharing all my passions and dreams. Words are not
enough to express how lucky, grateful and overwhelmed I am for being your soul
mate. I dedicate this thesis to you and our little Joel with all my love.
In Helsinki, January 2013
You
73
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