cellular cholestorol accumulation and egress from
TRANSCRIPT
Helsinki University Biomedical Dissertations No. 120
Cellular Cholesterol Accumulation and Egress from
Endosomal Compartments
Matts D. Linder
Institute of Biomedicine,
University of Helsinki
Finland
Academic Dissertation
To be publicly discussed with the permission of the Medical Faculty of the University of Helsinki,
at the Women’s clinic, Helsinki University Central Hospital, Haartmaninkatu 2,
big lecture hall, on April 24th 2009, at 12 o’clock noon.
Helsinki 2009
2
SUPERVISOR: Professor Elina Ikonen Institute of Biomedicine University of Helsinki Helsinki Finland REVIEWERS: Professor Markku Savolainen Department of Internal Medicine University of Oulu Oulu Finland Docent Jussi Jäntti Institute of Biotechnology University of Helsinki Helsinki Finland OPPONENT: Professor Arnold von Eckardstein Department for Clinical Chemistry University Hospital Zurich Zurich Switzerland ISBN 978-952-10-5469-3 (paperback) ISBN 978-952-10-5470-9 (PDF) ISSN 1457-8433 http://ethesis.helsinki.fi Helsinki 2009 Yliopistopaino
4
TABLE OF CONTENTS
ORIGINAL PUBLICATIONS.......................................................................................................... 6
ABBREVIATIONS............................................................................................................................. 7
INTRODUCTION............................................................................................................................... 9
REVIEW OF THE LITERATURE ...............................................................................................11 1. PATHOPHYSIOLOGY OF ATHEROSCLEROSIS ...............................................................11 1.1 Atherogenic lipoproteins and macrophage foam cell formation ...........................................11 1.2 Macrophage reverse cholesterol transport ..............................................................................12
1.2.1 ABCA1 mediated lipid efflux ...............................................................................13
2. INTRACELLULAR CHOLESTEROL TRAFFICKING AND HOMEOSTASIS................15 2.1 Uptake and processing of LDL cholesterol in the endosomal system ..................................15 2.2 Regulation of cholesterol biosynthesis in the ER...................................................................16 2.3 Transcriptional regulation of cholesterol levels......................................................................17 3. NIEMANN-PICK TYPE C DISEASE......................................................................................18 3.1 Disease pathophysiology..........................................................................................................18 3.2 The NPC1 and NPC2 proteins .................................................................................................20
3.2.1 Structural features and sterol binding of NPC1.................................................20
3.2.2 Subcellular localization and trafficking of NPC1 ..............................................22
3.2.3 Structural features and lipid binding of NPC2...................................................22
3.2.4 Subcellular localization and trafficking of NPC2 ..............................................25
4. RAB GTPases IN MEMBRANE AND CHOLESTEROL TRAFFICKING .........................26 4.1 Rab GTPase function................................................................................................................26 4.2 Rab proteins in intracellular sterol trafficking ........................................................................27 4.3 The Rab8 GTPase.....................................................................................................................28
AIMS OF THE STUDY ...................................................................................................................31
METHODS.........................................................................................................................................32
RESULTS AND DISCUSSION ......................................................................................................33
1. Defective intracellular trafficking of NPC proteins in NPC disease (I) ...............................33 1.1 Effects of individual mutations on the NPC1 protein ............................................................33 1.2 Intracellular localization and regulation of the NPC2 protein in NPC1 cells.......................35
2. Complementation of Niemann-Pick type C disease by Rab overexpression (II)..............36 2.1 Use of filipin staining as a measure of cholesterol efflux from endosomal compartments .37 2.2 Rab8 restores sphingolipid trafficking in NPC fibroblasts ....................................................38 2.3 Rab8 is resistant to membrane retention during cholesterol loading ....................................38 2.4 Rab8 siRNA induces a cholesterol accumulation phenotype ................................................39
3. Rab8 regulates cholesterol processing in primary human macrophages (III)....................40
5
3.1 Rab8 is expressed in macrophages present in atherosclerotic lesions in vivo......................40 3.2 Adenoviral overexpression of Rab8 in vitro increases ABCA1 dependent cholesterol efflux to apoA-I..........................................................................................................................................40 3.3 Rab8 RNAi inhibits cholesterol efflux and regulates ABCA1 trafficking in primary human macrophages....................................................................................................................................41
CONCLUSIONS AND FUTURE PROSPECTS..........................................................................44
ACKNOWLEDGEMENTS.............................................................................................................48
REFERENCES..................................................................................................................................50
6
ORIGINAL PUBLICATIONS This thesis is based on the following articles, which are referred to in the text by their roman
numerals.
I Blom TS, Linder MD, Snow K, Pihko H, Hess MW, Jokitalo E, Syvänen AC and
Ikonen, E. Defective endocytic trafficking of NPC1 and NPC2 underlying infantile
Niemann-Pick type C disease. Hum Mol Genet. 2003 Feb 1;12(3):257-72.
II Linder MD, Uronen RL, Hölttä-Vuori M, van der Sluijs P, Peränen J and Ikonen E.
Rab8-dependent recycling promotes endosomal cholesterol removal in normal and
sphingolipidosis cells. Mol Biol Cell. 2007 Jan;18(1):47-56.
III Linder MD, Mäyränpää MI, Peränen J, Pietilä TE, Pietiäinen VM, Uronen RL,
Olkkonen VM, Kovanen PT and Ikonen E. Rab8 Regulates ABCA1 Cell Surface
Expression and Facilitates Cholesterol Efflux in Primary Human Macrophages.
Arterioscler Thromb Vasc Biol. 2009 Mar 19. [Epub ahead of print]
7
ABBREVIATIONS
aa amino acid
ABCA1 ATP-binding cassette transporter A1
ACAT acyl-coenzyme A: cholesterol acyltransferase
acLDL acetylated LDL
apoA-I apolipoprotein A-I
BODIPY boron-dipyrromethene
ER endoplasmic reticulum
GDI guanosine nucleotide dissociation inhibitor
GSL glycosphingolipid
HDL high density lipoprotein
HMGR 3-hydroxy-3-methylglutaryl-CoA reductase
Insig Insulin-induced gene
LacCer lactosylceramide
Lamp lysosome-associated membrane protein
LBPA/BMP lysobisphosphatidic acid/bismonoacylglycerophosphate
LDL low density lipoprotein
LDLr low density lipoprotein receptor
LPDS lipoprotein-deficient serum
LAL lysosomal acid lipase
LXR liver X receptor
MDCK Madin-Darby canine kidney
mmLDL minimally modified LDL
MPR mannose-6-phosphate receptor
M6P mannose-6- phosphate
mRNA messenger RNA
NPC Niemann-Pick type C
NPC1 Niemann-Pick C 1 protein
NPC2 Niemann-Pick C 2 protein
NPC1L1 Niemann-Pick C 1 –like 1 protein
8
oxLDL oxidated LDL
PM plasma membrane
RCT reverse cholesterol transport
RNA ribonucleic acid
SCAP SREBP cleavage activating protein
siRNA small interfering RNA
SR-A1 scavenger receptor A1
SREBP sterol regulatory element binding protein
SSD sterol-sensing domain
TGN trans-Golgi network
TfR transferrin receptor
7DHCR 7-dehydrocholesterol reductase
WB Western blot
9
INTRODUCTION
Intracellular cholesterol homeostasis is a tightly regulated process aimed at balancing the flux of
cholesterol entering and exiting the cell. Defects in cellular cholesterol metabolism have been
linked to a variety of diseases ranging from atherosclerosis to Alzheimer’s disease. The
pathogenesis of these diseases are, however, quite complex, and it has been challenging to
pinpoint the exact cellular processes that lead to disease progression, although the relationship
between circulating cholesterol levels and atherosclerosis has been well documented. In general,
LDL particles in the circulation are clinically regarded as “bad cholesterol” since these particles
get entrapped in the vascular wall, leading to atherosclerosis. Circulating HDL particles are
conversely regarded as “good cholesterol” because of their ability to transport cholesterol from
peripheral tissues to the liver for secretion as bile salts.
During the progression of atherosclerosis, LDL cholesterol accumulates in the wall of arteries,
especially in places with high shear stress. Once inside the vessel intima, the LDL particles
undergo a series of chemical modifications either by enzymatic processes or through oxidation.
The modified LDL particles are then engulfed by macrophages, resulting in macrophage foam
cells. If the macrophage foam cells are not able to efflux the cholesterol back into the
bloodstream, the excessive cholesterol ultimately leads to cell death, and the deposition of
cellular debris within the atherosclerotic lesion. The cells ability to secrete cholesterol is mainly
dependent on the ABCA1 transporter which transfers cholesterol to extracellular apoA-I
particles, yielding nascent HDL particles. The nascent HDL particles are then further enriched
with cholesterol by the ABCG1 transporter. The process of atherosclerotic plaque development is
therefore to a large extent a cellular one, in which the capacity of the macrophages in handling
the excessive cholesterol load determines the progression of lesion development. Hence it is of
great importance to understand how cholesterol is transported within the cell. To achieve this we
have utilized the autosomal recessive cholesterol transport disease Niemann-Pick type C (NPC),
as a cellular model and tool to study the processes governing intracellular sterol transport.
In the first part of the study we characterized the mutations in an NPC1 disease patient and
studied the consequences of the individual mutations on the function of the NPC1 protein. A
10
major finding was that mutations in the NPC1 protein cause an upregulation of NPC2 protein
gene transcription, lending further support to the theory that these two proteins might function in
concert to facilitate cholesterol egress from the endosomal system.
In the second part of the study we used NPC1 disease cells as a cellular model for cholesterol
accumulation, in order to identify individual Rab GTPases involved in cholesterol efflux from the
endosomal system. Using a combination of fluorescent microscopy and biochemical techniques
we identified Rab8 as a key protein in facilitating the transport of endocytosed LDL cholesterol
to extracellular acceptors. Knock-down of the Rab8 protein with RNAi resulted in intracellular
cholesterol accumulation, while overexpression of Rab8 alleviated the cholesterol storage
phenotype seen in NPC1 disease cells.
In the third part of the study we studied the role of Rab8 in macrophage foam cell formation and
cholesterol processing. We found that Rab8 is expressed in macrophage foam cells of
atherosclerotic lesions. Overexpression of Rab8 in primary human macrophages induced
ABCA1-mediated cholesterol efflux to apoA-I. Rab8 facilitated the efflux of cholesterol from
macrophage foam cells by regulating ABCA1 protein levels at the cell surface, as well as
facilitating cholesterol traffic to ABCA1 substrate pools.
REVIEW OF THE LITERATURE
1. PATHOPHYSIOLOGY OF ATHEROSCLEROSIS
1.1 Atherogenic lipoproteins and macrophage foam cell formation Although several proatherogenic factors have been identified, the level of circulating LDL
particles stands out as the most important determinant of lesion development. Atherosclerotic
plaques start out as fatty streaks in the intima of middle and large diameter arteries already at
early adulthood. The process of plaque formation is complex but lesions usually form at places
with high shear stress. LDL is deposited in the intimal matrix of the artery where it is modified to
yield so called minimally modified LDL (mmLDL) and oxidized LDL (oxLDL) (Schwenke and
Carew, 1989; Williams and Tabas, 1998). MmLDL can still be recognized by LDL receptors
while oxLDL is no longer recognized (Navab et al., 1996). Instead, oxLDL is taken up by
macrophages via different receptors, the most important of which are CD36 and scavenger
receptor A1 (SR-A1). The lesional macrophages are derived from infiltrating monocytes that are
attracted to the site by various chemotactic factors, which can be secreted by other cells in the
lesion, or can be components of oxLDL, e.g. lyso-phosphatidylcholine (lyso-PtdCho) (Hansson
and Libby, 2006; Quinn et al., 1987).
Blood-borne monocytes infiltrate the arterial intima and differentiate into macrophages, which
take up modified LDL particles. This is initially thought to serve as an atheroprotective function.
The internalized cholesterol is stored as cholesterol esters in cytoplasmic lipid droplets.
Macrophages are, however, not capable of downregulating the scavenger receptors on the plasma
membrane, which leads to continued scavenging of lipoprotein particles and expanding
intracellular cholesterol stores, giving a foamy appearance of the cytoplasm. The foam cells are
not able to efflux cholesterol at the same rate as uptake, leading to cell death, and the deposition
of cellular debris in the arterial intima (Figure 1).
MATTS LINDER
12
Figure 1 Macrophage foam cells in atherosclerotic lesion development. Circulating LDL
particles accumulate in the intima of arteries, and undergo chemical modification, resulting in
oxidized LDL (oxLDL). Blood-borne monocytes infiltrate the area and differentiate into
macrophages that scavenge the oxLDL particles. Macrophage foam cells undergo apoptosis
which results in a necrotic core, leading to further lesion development. Modified from (Glass and
Witztum, 2001).
1.2 Macrophage reverse cholesterol transport
Reverse cholesterol transport (RCT) is the process of net cholesterol flux from peripheral tissues
to the liver (Cuchel and Rader, 2006). In terms of atherosclerosis, the process of macrophage
RCT warrants the most attention, although conceivably RCT might also take place from other
cells in the atherosclerotic lesion. In terms of macrophage RCT, the combined role of the ATP-
binding cassette transporters ABCA1 and ABCG1 seem to be the most important. ABCA1 was
found to be mutated in Tangier disease (Bodzioch et al., 1999; Brooks-Wilson et al., 1999; Rust
et al., 1999) Although SR-B1 has also been implicated in cholesterol efflux in vitro it is not
involved in macrophage RCT in the mouse (Wang et al., 2007).
REVIEW OF THE LITERATURE
13
1.2.1 ABCA1 mediated lipid efflux The primary function of ABCA1 is thought to be the lipidation of apolipoprotein A-I (apoA-I) at
the plasma membrane (Denis et al., 2008), although intracellular compartments may also be
involved (Cavelier et al., 2006; Hassan et al., 2008; Neufeld et al., 2004). However, the
contribution of intracellular apoA-I to total HDL production has been estimated to be only ~1.4%
of total HDL production (Denis et al., 2008). The precise mechanism of apoA-I lipidation is not
known. It has been shown in numerous studies that apoA-I interacts directly with ABCA1
(Chroni et al., 2004; Vedhachalam et al., 2004) and that the binding is essential for ABCA1
mediated lipid transfer to ABCA1 (Fitzgerald et al., 2002). However, there seems to be two
separate binding sites for apoA-I on the plasma membrane, a low- capacity binding site, thought
to represent direct binding to ABCA1, and a high-capacity binding site to membrane domains
generated through ABCA1 action (Hassan et al., 2007; Vedhachalam et al., 2007). The binding of
apoA-I to ABCA1 is relatively unspecific and apparently involves amphiphatic alpha-helices in
apoA-I (Fitzgerald et al., 2002; Vedhachalam et al., 2007). Accordingly, N- and C-terminal
domain mutants of apoA-I bind equally well to ABCA1. In contrast, insertion of apoA-I into the
plasma membrane domains requires intact hydrophobic C-terminal domain alpha-helices (Gillotte
et al., 1999; Saito et al., 2003; Saito et al., 2004). The lipidation process might involve the
transfer of phospholipids to apoA-I, generating nascent HDL particles, a process which is
defective in Tangier disease patients (von Eckardstein et al., 1998). ABCA1 might function by
flipping of phospholipid species from the inner to the outer leaflet (Alder-Baerens et al., 2005),
resulting in membrane bending and increased curvature, facilitating the binding and subsequent
lipidation of lipid poor apoA-I at the plasma membrane (Vedhachalam et al., 2007).
Binding of apoA-I to ABCA1 might initiate signaling responses via the Janus kinase 2,
stabilizing the protein on the cell surface (Tang et al., 2006; Tang et al., 2004). ABCA1 and
CDC42 are in close proximity of each other at the plasma membrane and Golgi compartment,
and these proteins co-immunoprecipitate (Tsukamoto et al., 2001). It was subsequently shown
that binding of apoA-I to ABCA1 also initiates signaling via CDC42 and phosphorylation of
PAK-1 and p54JNK, leading to actin polymerization (Nofer et al., 2006). There is evidence that
apoA-I interacts directly with ABCA1 since these proteins cross-link, indicating that they are in
close proximity (<7 Å) of each other (Chroni et al., 2004; Denis et al., 2004; Fitzgerald et al.,
MATTS LINDER
14
2004; Wang et al., 2000). Additionally, binding of apoA-I enhances the interaction between
ABCA1 and CDC42 (Nofer et al., 2006)
REVIEW OF THE LITERATURE
15
2. INTRACELLULAR CHOLESTEROL TRAFFICKING AND HOMEOSTASIS
2.1 Uptake and processing of LDL cholesterol in the endosomal system Regulation of cellular cholesterol homeostasis is mainly achieved through balancing the amount
of cholesterol uptake, synthesis and efflux. Circulaing LDL particles containing cholesterol bind
to the LDL receptor at the cell surface. The LDL receptor complex is then internalized through
clathrin-coated pits. Once endocytosed, the LDL particles dissociate from the LDL receptor
which then recycles back to the cell surface (Matter et al., 1993). Cholesteryl esters in the LDL
particle are then hydrolyzed by lysosomal acid lipase (LAL) to yield free (unesterified)
cholesterol. Most of the cholesterol in the endocytic compartments is present in multivesicular
endosomes (multivesicular bodies, MVB) and recycling endosomes, ~60% and ~20%
respectively (Möbius et al., 2003). The internal membranes of MVBs contain the bulk of
cholesterol and are enriched in the endosome specific phospholipid LBPA/BMP
(lysobisphosphatidic acid / bismonoacyl-glycerophosphate) (Kobayashi et al., 1999). The free
cholesterol exits the endosomal compartments in a process that is dependent on the NPC1 and
NPC2 proteins. After exiting the endosomes, cholesterol is mainly distributed to the plasma
membrane and Golgi apparatus by yet unidentified pathways, before finally reaching the ER. The
ER is usually relatively cholesterol poor and cholesterol entering the ER is rapidly converted to
cholesteryl esters by acyl-coenzyme A: cholesterol acyltransferase (ACAT), for storage in lipid
droplets (Figure 2).
MATTS LINDER
16
Figure 2. Cholesterol uptake and compartmentalization. LDL particles bind to the LDL-receptor
at the plasma membrane, and are endocytosed through clathrin-coated pits. The LDL particle
then dissociates from the LDLr which is recycled back to the plasma membrane. The cholesterol
esters derived from the LDL particle is hydrolyzed in the endosomes to yield free cholesterol. The
free cholesterol is transported out of the endosomal compartments to the plasma membrane and
TGN before reaching the ER (dotted arrows). In the ER, the cholesterol is re-esterified by ACAT,
and stored in lipid droplets. (LDLr, LDL receptor; EE, early endosome; RE, recycling
endosome; MVB, multi vesicular body; LE, late endome; LY, lysosome; ER, endoplasmic
reticulum; LD, lipid droplet; TGN trans-Golgi network; N, nucleus )
2.2 Regulation of cholesterol biosynthesis in the ER
The ER is the main orchestrator of cellular cholesterol distribution and amount. However, the ER
is a relatively cholesterol poor organelle with a cholesterol content of only 1% of total cellular
cholesterol (Lange, 1991). The two main proteins mediating cholesterol regulation in the ER are
REVIEW OF THE LITERATURE
17
HMG-CoA reductase (HMGR) and the Sterol response element binding protein (SREBP)
Cleavage Activating Protein (SCAP), which forms a complex with SREBP in the ER (Goldstein
et al., 2006). HMGR is the rate-limiting protein in de novo cholesterol biosynthesis, while SCAP
regulates SREBP processing. Both proteins are regulated by sterol binding to the sterol-sensing
domain (SSD) and the ER resident Insig proteins (Insig1 and Insig2). Rising cholesterol levels in
the ER induces regulatory mechanisms at both transcriptional and posttranscriptional levels.
Cholesterol binds directly to the SSD of SCAP, causing a conformational change in the SSD.
This causes SCAP to bind to Insig proteins, leading to retention of the SCAP/SREBP complex in
the ER, thus shutting down SREBP dependent gene transcription (Yang et al., 2002). HMGR is
also regulated by sterols, but the outcome of Insig binding is entirely different. Binding of
HMGR to Insigs leads to ubiquitinylation and subsequent degradation of HMGR in the
proteasome, thus reducing de-novo cholesterol synthesis (Espenshade and Hughes, 2007; Sever
et al., 2003).
2.3 Transcriptional regulation of cholesterol levels
There are two main transcriptional regulatory systems in the cell which work antagonistically to
balance the intake, de novo synthesis and efflux of cholesterol, namely the sterol regulatory
element proteins (SREBPs) and liver X receptors (LXRs). As described above, the SREBP
system is regulated by ER sterol levels through the SCAP protein. The role of SCAP is to
regulate the trafficking of SREBP to the Golgi. Upon cholesterol depletion in the ER, SREBP
traffics to the Golgi where it is activated through a proteolytic process by the site1 (S1P) and
site2 (S2P) proteases. The active transcription factor travels to the nucleus where it activates the
transcription of gene products which increase intracellular cholesterol levels, e.g. HMGR and the
LDL receptor (Goldstein et al., 2006). Conversely, activation of LXRs is mediated by oxysterols
leading to the transcription of gene products that facilitate cholesterol efflux from the cell, e.g.
the ABCA1 transporter (Tontonoz and Mangelsdorf, 2003).
MATTS LINDER
18
3. NIEMANN-PICK TYPE C DISEASE
3.1 Disease pathophysiology
Niemann-Pick type C disease is a severe lysosomal cholesterol-sphingolipid storage disorder.
The progress of the disease is varying with onset of symptoms ranging from early infancy to
adulthood. Typically, the disease presents with neurological regression and peripheral
manifestations due to massive cholesterol accumulation in the liver and spleen. Clinically the
patients present with hepatosplenomegaly, ataxia, dystonia, seizures, supranuclear gaze palsy,
and progressive dementia. Early-onset NPC disease is usually more severe and involves
peripheral tissues with hepatosplenomegaly often observed. In the brain, there is degeneration of
the brain stem and Purkinje neurons of the cerebellum (Walkley and Suzuki, 2004). Why only
parts of the brain and specific subsets of neurons are affected is not understood, but the process is
likely to involve both apoptosis and autophagy (Ko et al., 2005; Pacheco et al., 2007). A number
of biochemical defects have been described, but the traditional hallmark of the disease is
cholesterol accumulation in lysosomes combined with decreased esterification of cholesterol in
the endoplasmic reticulum (ER) (Blanchette-Mackie et al., 1988). NPC has therefore historically
been regarded as a cholesterol transport disorder. This is in contrast to Niemann-Pick types A and
B, which are caused by acid sphingomyelinase deficiency. Interestingly, there is concomitant
cholesterol accumulation in Niemann-Pick type A and B cells, and the depletion of cholesterol
from these cells restores trafficking of fluorescently tagged lactosyl ceramide (BODIPY-LacCer)
(Puri et al., 1999). These results emphasize the interdependence of cholesterol and sphingolipids
in lysosomal storage disorders. Accordingly, in NPC disease there is a concomitant sphingolipid
accumulation and decreased sphingomyelinase activity (Liscum and Klansek, 1998; Lloyd-Evans
et al., 2008). The pathophysiology of NPC disease is however quite complex and it is has been
challenging to pinpoint the precise pathological mechanism. Nevertheless it is likely that the
disease phenotype is the combined result of aberrant cholesterol and sphingolipid metabolism.
REVIEW OF THE LITERATURE
19
Figure 3. Primary human fibroblasts from control and NPC1 patients. Cells are stained with
filipin which visualizes free cholesterol (blue). NPC1 cells accumulate large amounts of
cholesterol perinuclearly.
One of the main cellular perturbations is a defect in the regulatory responses to cholesterol
loading via the LDL pathway. Normally, cholesterol containing LDL particles taken up via the
LDL-receptor, where after the cholesterol exits the endosomal system and is transported to the
plasma membrane and to the ER, possibly via the trans-Golgi network (TGN). Superfluous
cholesterol is then re-esterified by the ER resident protein acyl-CoA acyltransferase (ACAT) for
storage in lipid droplets (Chang et al., 2001). In NPC disease cholesterol is trapped in aberrant
endosomes and fails to reach the ER (Blanchette-Mackie et al., 1988; Pentchev et al., 1985)
(Figure 3). This results in a relative cholesterol deficit in the ER, which is the primary sterol-
sensing organelle in the cell. As a result, there is a misregulation of the two major cholesterol
homeostasis regulatory machineries i.e. the SREBP and LXR pathways. Thus, de novo
cholesterol synthesis is not down-regulated, but rather increased (Liscum and Faust, 1987). Also
the cholesterol efflux pathways are not up-regulated appropriately, and the LDL-receptor fails to
down-regulate. The failure of down-regulation of the SREBP pathway can easily be attributed to
the relative lack of cholesterol in the ER. Concomitantly, there is defective generation of
oxysterols, the key activators of the LXR pathway (Frolov et al., 2003).
Control NPC1
MATTS LINDER
20
3.2 The NPC1 and NPC2 proteins
Niemann-Pick disease type C is comprised of two separate genetic complementation groups with
mutations in either of the NPC1 or NPC2 genes. Roughly 95% of disease cases are caused by
mutations in the NPC1 gene whilst the remaining cases are accounted for by mutations in the
NPC2 gene (Carstea et al., 1997; Naureckiene et al., 2000). The precise function of the two NPC
proteins has not been established. However, lack of either protein leads to a nearly identical
cellular phenotype, indicating that these proteins must perform similar functions in sterol egress
from the endosomal system. Also, the NPC2/NPC1 double knockout mouse model displays an
identical phenotype to that of NPC2 and NPC1 knockout models alone (Sleat et al., 2004). NPC2
is a cholesterol binding protein present in lysosomes. NPC1 has been suggested to function as a
transmembrane fatty acid transporter (Davies et al., 2000a), and more recently, as a sphingosine
transporter (Lloyd-Evans et al., 2008). The conclusive evidence for a specific transport function
for either of the proteins is however still lacking.
3.2.1 Structural features and sterol binding of NPC1 The gene mutated in the major complementation group was identified in 1997 through a
positional cloning strategy. The gene product, named NPC1, was found to be a large, 1278 amino
acid polytopic membrane protein with 13 transmembrane spans (Carstea et al., 1997) (Figure 4).
A number of interesting domains have been identified in the protein, including a putative sterol-
sensing domain (SSD) comprising of transmembrane loops three through seven (Davies and
Ioannou, 2000). This domain was originally identified in the SCAP (sterol regulatory element
binding protein (SREBP) cleavage activating protein) and hydroxymethylglutaryl (HMG)-CoA
reductase proteins (HMGR) (Osborne and Rosenfeld, 1998). Other proteins containing a SSD
include the Hedgehog morphogen receptor Patched and 7-Dehydrocholesterol reductase
(7DHCR) (Kuwabara and Labouesse, 2002), as well as Niemann-Pick type C 1 Like 1 (NPC1L1)
(Davies et al., 2000b). The function of the SSD has most extensively been studied in SCAP and
HMGR. The SSD is required for cholesterol dependent retention of SCAP in the ER, as well as
lanosterol-induced ubiquitinylation and degradation of HMGR (Nohturfft et al., 1999; Song et al.,
2005). The evidence regarding a specific sterol-sensing function of the SSD in the NPC1 protein
REVIEW OF THE LITERATURE
21
is still inconclusive. NPC1 is capable of binding to a photoactivatable cholesterol analog,
azocholestanol, while mutations in the SSD (P691S, Y634C) abolish this binding (Ohgami et al.,
2004). In vitro [3H]-cholesterol binding to the purified SSD of SCAP has also been established,
but this was not inhibited by the corresponding Y298C mutation in SCAP (Radhakrishnan et al.,
2004). However this mutation in SCAP has previously been shown to inhibit the binding of
SCAP to Insig proteins involved in 25-hydroxycholesterol sensing in the ER (Radhakrishnan et
al., 2007; Yang et al., 2002). Also, mutations in NPC1, corresponding to gain of function
mutations in SCAP, resulted in increased cholesterol trafficking in the cell (Millard et al., 2005).
However, in a later study the sterol binding capacity of the NPC1 protein was limited to the N-
terminal domain (“NPC domain”) of purified NPC1 (Infante et al., 2008a). Mutations in the SSD
are none the less clinically significant, and mutations in the SSD abolish the cholesterol transport
function of NPC1 (Watari et al., 1999b). A critical role for the N-terminal domain in NPC1
function has also previously been shown, as well as for the C-terminal LLNF lysosome-targeting
motif (Watari et al., 1999b). Deletion of the C-terminal dileucine motif resulted in the retention
of the NPC1 protein in the ER. Site directed mutagenesis of cysteins (C63S, C74S/C75S and
C97S) resulted in non-functional NPC1 protein that localized to the surface of lysosomes (Watari
et al., 1999a).
Figure 4. Schematic representation of the NPC1 protein, The SSD is shown in red. Adapted from
(Davies and Ioannou, 2000).
MATTS LINDER
22
3.2.2 Subcellular localization and trafficking of NPC1 The NPC1 and NPC2 proteins are thought to reside mainly in late endocytic compartments. The
proteins show little co-localization, indicating that they are present in different subsets of
endosomes. NPC1 colocalizes with the late endosomal/lysosomal marker Lamp2, but not with
endocytosed LDL derived cholesterol or the mannose-6- phosphate receptor (MPR) (Neufeld et
al., 1999). NPC1 has also been shown to colocalize with LBPA, Rab7, and Rab9 (Higgins et al.,
1999; Puri et al., 1999; Zhang et al., 2001b). Drugs that induce lysosomal cholesterol
accumulation e.g. progesterone and U18666A redistributes NPC1 to cholesterol laden lysosomes
(Neufeld et al., 1999). In normal cells NPC1 is thought to only transiently localize to cholesterol
enriched organelles (Higgins et al., 1999; Kobayashi et al., 1999; Neufeld et al., 1999; Patel et al.,
1999; Watari et al., 1999a; Zhang et al., 2001b). Also the GM2 ganglioside localizes exclusively
with NPC1, but not with endocytosed cholesterol (Osborne and Rosenfeld, 1998).
Live cell microscopy using GFP-tagged NPC1 protein has revealed that NPC1 containing
endosomes are highly mobile. NPC1 function seems to be dually required for both lipid sorting
and tubular late endocytic trafficking (Ko et al., 2001). Also the motility of NPC1 containing
endosomes is modulated by the lipid composition of the membranes, and is inhibited by
cholesterol accumulation (Lebrand et al., 2002; Zhang et al., 2001a). NPC1-GFP containing
organelles undergo tubulation and fission with both anterograde and retrograde migrations along
microtubules. This movement also requires an intact SSD (Zhang et al., 2001a). Newly
synthesized NPC1-GFP was also seen by live cell microscopy to be transported from the ER via
the Golgi to late endosomes (Zhang et al., 2001a). NPC1 is ubiquitinylated upon cholesterol
depletion which might influence the trafficking of NPC1 within different subsets of endosomes
(Ohsaki et al., 2006).
3.2.3 Structural features and lipid binding of NPC2 The NPC2 gene product is a 151 amino-acid soluble lysosomal protein containing a 19 amino
acid signal peptide which is cleaved off to generate the active 132 amino-acid protein.
REVIEW OF THE LITERATURE
23
Intracellular localization studies have shown that the protein resides both in Lamp1 positive and
Lamp1 negative endosomes, as well as the Golgi apparatus. The NPC2 protein is glycosylated
and contains a mannose-6-phosphate (M6P) post-translational modification conferring lysosomal
targeting through binding to the mannose-6-phosphate receptor (MPR). The NPC2 protein was
originally characterized as a cholesterol binding protein found in epididymal fluid named HE1
(human epididymis protein 1) (Okamura et al., 1999). As a result of a screening for M6P
containing proteins HE1 was found to localize to lysosomes. This allowed the identification of
HE1 as the second protein in NPC disease, and the HE1 protein was renamed NPC2
(Naureckiene et al., 2000).
Structural analysis of the bovine NPC2 protein and site-directed mutagenesis strategies have
shown the mechanism of cholesterol binding and identified functional domains in the protein.
The bovine orthologue of NPC2, EPV20 had previously been purified from bovine milk (Larsen
et al., 1997). The structure of the bovine homolog bNPC2 was elucidated by x-ray
crystallography, revealing an Ig-like -sandwich fold consisting of seven -strands arranged in
two -sheets (Friedland et al., 2003). Between the -sheets, three small cavities are formed with a
total volume of 158 Å3. The volume of a cholesterol molecule is 741 Å3, indicating that the -
sandwich needs to expand in order to accommodate the cholesterol molecule. The structure of
NPC2 bound to cholesterol-3-O-sulfate also supports this model (Xu et al., 2007). X-ray
crystallography of NPC2 bound to cholesterol sulfate indicates that the sterol is inserted into the
cholesterol binding pocket with the iso-octyl side chain inside the pocket, and the sulfate group at
position 3 of the A ring protruding from the entrance (Xu et al., 2007). Such an orientation would
explain why NPC2 has low affinity for oxysterols (Infante et al., 2008a). A mutational screen
based on evolutionally conserved residues highlights the importance of the cholesterol-binding
cavity (Ko et al., 2003) (Figure 5).
MATTS LINDER
24
Figure 5. Structure of the NPC2 sterol binding pocket. (A) Stereo view of the proposed sterol
binding cavity. Sidechains of residues lining the cavity are shown in stick representation. Side
chains required for cholesterol binding (Ko et al., 2003) are shown in red. (B) Space filling
model of cholesterol (geeen) docked in the proposed sterol-binding site. Reprinted from
(Friedland et al., 2003) with the permission of the publisher. Copyright (2003) National Academy
of Sciences, U.S.A.
The current evidence strongly favors a direct cholesterol shuttling model for NPC2 function.
Several studies have now established that NPC2 binds cholesterol (Friedland et al., 2003; Infante
et al., 2008a; Ko et al., 2003; Liou et al., 2006). NPC2 is able to extract and insert cholesterol
from membranes in vitro (Cheruku et al., 2006) as well as shuttle cholesterol between liposomal
vesicles (Babalola et al., 2007). NPC2 can also transfer cholesterol between the N-terminal
domain of NPC1 and liposomes in vitro (Infante et al., 2008b). NPC2 is also involved in Thymal
T cell selection by loading the MHC class I-like protein CD1d with the selecting
glycosphingolipid isoglobotrihexosylceramide (iGb3) in NKT cells (Schrantz et al., 2007).
Whereas the in vitro loading of iGb3 to CD1d required dimerization of NPC2, the transfer was
not affected by cholesterol binding.
REVIEW OF THE LITERATURE
25
3.2.4 Subcellular localization and trafficking of NPC2 The NPC2 protein resides mainly in the lumen of late endosomes/lysosomes. The intracellular
trafficking of NPC2 is governed by the mannose 6-phosphate receptor (MPR) owing to the
posttranslational addition of a mannose 6-phosphate moiety to the protein (Naureckiene et al.,
2000). The mannose 6-phosphate moiety is recognized by the MPR, which enables trafficking
between the Golgi apparatus and lysosomes as well as uptake from the PM. Depletion of the two
MPRs, MPR300 (cation-independent) and MPR46 (cation-dependent), in fibroblasts increases
the secretion of NPC2 from cells (Willenborg et al., 2005). NPC2 is abundantly present in
epididymal fluid and milk (Kirchhoff et al., 1996; Larsen et al., 1997), and can be secreted from
cells in the cholesterol bound form (Ko et al., 2003; Okamura et al., 1999). NPC2 is also secreted
from cultured glia cells, but does not associate with the major secretory sterol containing
lipoprotein particles. Also, overexpression of NPC2 in glia cells does not enhance sterol secretion
(Mutka et al., 2004). Secreted NPC2 can be re-endocytosed and is biologically active. Incubation
of NPC2 cells with medium preconditioned by incubation with WT cells complements the
cholesterol storage phenotype, but the complementation is inhibited by mannose-6 phosphate,
indicating that endocytosis by MPR is required (Naureckiene et al., 2000). However
overexpression of NPC2 mutants that are not properly secreted still complement the individual
transfected cells, indicating that secretion is not an absolute necessity (Ko et al., 2003).
NPC2 is glycosylated, appearing as two individual bands of 21 to 25 kDa in WB of human
fibroblast samples. After removal of N-linked glycans with PNGaseF (protein:N-glycosidase F)
the protein migrates at 18 kDa (Mutka et al., 2004). A mutational screen of the possible
asparagine linked (Asn, N) glycosylation sites revealed that only two of the sites, N58 and N135,
are utilized. The study also showed that glycosylation of Asn 58 is required for proper lysosomal
targeting of NPC2 (Chikh et al., 2004).
MATTS LINDER
26
4. RAB GTPases IN MEMBRANE AND CHOLESTEROL TRAFFICKING
4.1 Rab GTPase function Rab proteins are small GTPases belonging to the Ras superfamily of GTPases (Wennerberg et al.,
2005). Rab proteins function as molecular switches, regulating events such as vesicle trafficking,
motility, docking and fusion (Pfeffer, 2001; Zerial and McBride, 2001). Rab proteins cycle
between the cytosol and target membranes in an active GTP-bound state and an inactive GDP-
bound state. The switching to the active state is mediated by guanine nucleotide exchange factors
(GEFs), which mediate binding of GTP to the Rab protein. Inactivation is mediated by GTPase-
activating proteins (GAPs) which catalyze the intrinsic GTPase activity of the Rab protein,
resulting in hydrolysis of the bound GTP to GDP (Pfeffer, 2001; Segev, 2001). In the active GTP
bound state, Rab proteins insert into the target membrane by virtue of a prenylation motif located
in the C-terminal of the protein. Prenylation of Rab proteins is facilitated by Rab escort protein
(REP) which presents newly synthesized Rab protein to Rab geranylgeranyl transferase
(GGTase) (Andres et al., 1993). In the ‘soluble’ state Rab proteins are bound and sequestered in
the cytosol by GDI (guanosine nucleotide dissociation inhibitor) (Figure 6). Human cells express
two isoforms, GDI which is enriched in the brain and the ubiquitously expressed GDI (Alory
and Balch, 2001). The binding of Rab proteins to GDI is strongly dependent on prenylation as
well as the presence of GDP in the nucleotide binding site. Upon GDP- or GTP binding the Rab
proteins change conformation in the highly conserved ‘switch region’, which is recognized by
GDI. GDI can also extract Rab proteins from membranes (Sasaki et al., 1990; Ullrich et al.,
1993).
The targeting of Rab proteins to specific membranes is thought to be mediated by the C-terminal
‘hypervariable domains’ which are the most divergent amino acid stretches between different
Rab proteins (Chavrier et al., 1991). The association of the Rab protein to GDI is probably
modulated by the length of the hypervariable domain with affinities of 20-500nM Kd being
reported (Schalk et al., 1996; Shapiro and Pfeffer, 1995). The deposition of Rab proteins into the
target membrane is facilitated by GDI-displacement factors (GDF).
REVIEW OF THE LITERATURE
27
Figure 6. The Rab cycle. Rab GTPases are activated by guanine nucleotide exchange factors
(GEFs). The Rab proteins then bind to effector proteins which can for instance be molecular
motors. After fulfilling its task, the Rab protein is deactivated by GTPase activating proteins
(GAPs), and removed from the membranes by guanine nucleotide dissociation inhibitor (GDI).
Another round of action is initiated by GDI displacement factor (GDF) which facilitates the
deposition of Rab proteins into the target membrane.
4.2 Rab proteins in intracellular sterol trafficking Cholesterol efflux from the endosomal compartments seems to be mediated, at least in part, by
Rab protein directed membrane trafficking. It was initially shown that inhibition of Rab protein
function by microinjection of Rab-GDI inhibits cholesterol removal from endocytic
compartments (Höltta-Vuori et al., 2000). So far, the individual Rab proteins that have been
implicated in intracellular cholesterol trafficking localize to the endocytic compartments of the
cell (Table 1). Choudhury and colleagues showed that overxpression of dominant negative Rab7
or Rab9 inhibited the Golgi targeting of sphingolipids, and that overexpression of wild-type Rab7
or Rab9 reduced the cholesterol accumulation in NPC1 fibroblasts (Choudhury et al., 2002).
MATTS LINDER
28
Rab9 was subsequently shown to increase cholesterol efflux in two separate studies (Narita et al.,
2005) (Walter et al., 2003). In a recent study, the life span of Rab9 / NPC1-/- transgenic mice was
also found to be increased by ~ 22 % as compared to NPC1-/- mice (Kaptzan et al., 2009). The
dominant negative form of Rab4 also perturbs cholesterol recycling (Choudhury et al., 2004).
Overexpression of Rab11 affects endosomal cholesterol trafficking, resulting in the accumulation
of free cholesterol in recycling endosomes (Höltta-Vuori et al., 2002).
Table 1. Localization and function of Rab proteins which have been shown to affect cholesterol
transport in the endocytic compartments.
Rab Localization Function Reference
Rab4 Early and recycling
endosomes
Rapid endosomal recycling (van der Sluijs et al., 1992)
Rab7 Late endosomes,
lysosomes
Early to late endosomal transport,
Lysosome biogenesis
(Press et al., 1998) (Bucci
et al., 2000)
Rab9 Late endosomes,
trans Golgi network
Late endosome to Golgi transport (Lombardi et al., 1993)
Rab11 Recycling endosomes Recycling through the endocytic
recycling compartment (ERC)
(Ullrich et al., 1996) (Ren et
al., 1998)
4.3 The Rab8 GTPase Two isoforms, Rab8A (referred to here as Rab8) and Rab8b have been identified in humans.
Rab8 is ubiquitously expressed in tissues while Rab8b is predominantly expressed in the spleen,
testis and brain (Armstrong et al., 1996). There is an 83% sequence identity between Rab8A and
Rab8b, with the highest degree of divergence within the C-terminal hypervariable domain (Figure
7) (Armstrong et al., 1996). Rab8 differs from other Rab proteins in that it is geranylgeranylated
at a single site as opposed to the usual double geranylgeranylation. This is due to the presence of
a CaaL motif instead of the usual CC or CXC motifs (Casey and Seabra, 1996; Wilson et al.,
1998).
REVIEW OF THE LITERATURE
29
Figure 7. Bar representation of Rab8A and Rab8b. Guanine base binding sites are indicated in
black (aa 33; aa 119-125; aa 147-154). The hypervariable domain (aa 154-207) is indicated in
yellow. The C-terminal prenylation motif CVLL is composed of aa 204-207.
Rab8 affects cell shape, inducing cellular protrusions when overexpressed (Nachury et al., 2007)
(Armstrong et al., 1996; Peränen et al., 1996). In two separate studies Rab8 was found to regulate
primary cilium formation (Nachury et al., 2007; Yoshimura et al., 2007). Cilium formation was
also found to be dependent on the Rab8 specific GAP XM_037557 (Yoshimura et al., 2007).
Polarized transport by Rab8 is thought to be partly mediated by reorganization of actin and
microtubules (Ang et al., 2003; Peränen et al., 1996). Rab8 also regulates the actin based
movement of melanosomes to the PM in MDCK cells (Chabrillat et al., 2005), as well as vesicle
transport during neuronal outgrowth. Knock-down of Rab8 with siRNA in neurons results in
inhibition of anterograde vesicle movement and disrupts neuronal outgrowth (Huber et al., 1995).
In addition to Rab8, vesicle formation and transport to membrane protrusions is dependent on the
Rab8 specific GDP/GTP exchange factor Rabin8 (Hattula et al., 2002). Rab8 was shown to
mediate apical protein transport in intestinal ephitelial cells in a Rab8 -/- mouse model. In
intestinal epithelial cells Rab8 might control both direct protein transport from the TGN to the
apical membrane, or indirectly via the basolateral membrane. In any case, Rab8 deficiency causes
the mislocalization of apical proteins to the lysosomes, and formation of intracellular microvillus
inclusions, resulting in dysfunction of apical microvilli and malabsorbtion (Sato et al., 2007).
Reduced Rab8 expression was also found in a human microvillus inclusion disease patient with
an identical ultrastructural phenotype to the Rab8-/- mouse (Sato et al., 2007). Recently it was
also established that mutations in the MYOB5 gene, which encodes the Rab8 interacting
myosinVb motor protein (Roland et al., 2007), also causes microvillus inclusion disease in
humans (Muller et al., 2008).
MATTS LINDER
30
Rab8 is involved in both constitutive transport of proteins from the Golgi (Huber et al., 1993) and
basolateral vesicle transport in Madin-Darby canine kidney (MDCK) cells (Ang et al., 2003). In
MDCK cells, recycling endosomes might serve as intermediates for Golgi to PM transport, and as
a common sorting organelle for the endocytic and secretory pathways (Ang et al., 2004). The
polarized transport of proteins to the basolateral membrane is dependent on the AP-1B clathrin
adaptor complex, which directs clatrin-coated bud formation at the TGN, and is regulated by
Rab8 (Ang et al., 2003). The Rho GTPase CDC42 also selectively regulates the AP-1B pathway,
and dominant negative CDC42 causes selective missorting of AP-1B dependent cargo, indicating
that Rab8 and CDC42 might function in the same pathway (Ang et al., 2003). In addition, cell
morphogenesis is also altered by the Rab8 interacting, TNF -inducable protein FIP-2
(optineurin), linking Rab8 to huntingtin (Hattula and Peränen, 2000). Optineurin also links Rab8
to myosin VI, which acts as a motor for actin dependent transport of vesicles deriving from the
TGN (Au et al., 2007; Sahlender et al., 2005). Mutant huntingtin (mhtt), in turn inhibits Rab8
mediated post-Golgi trafficking, by delocalizing the optineurin/Rab8 complex from the Golgi
(Del Toro et al., 2009).
Table 2. Proteins which have been shown to interact directly with Rab8.
Protein Function Reference
Rabin8 Rab8 specific GEF (Hattula et al., 2002)
XM_037557 Rab8 specific GAP (Yoshimura et al., 2007)
Myosin Vb Molecular motor (Roland et al., 2007)
Rabaptin5 Regulator of endocytosis (Omori et al., 2008)
FIP-2/optineurin Vesicle trafficking (Hattula and Peränen, 2000)
31
AIMS OF THE STUDY
The aim of the first part of the study was to gain insight in to the function of the NPC1 and NPC2
proteins. Making use of a severe form of infantile NPC1 disease the goal was to identify
individual disease causing mutations in the NPC1 protein and to study the consequences of the
individual mutations at the cellular level. The other key goal of the study was to establish the
intracellular location of the NPC2 protein in normal and NPC1 fibroblasts in order to gain further
insight into the pathophysiology of NPC disease.
The aim of the second part of the study was to use the cholesterol accumulation found in NPC1
disease as a tool to identify proteins involved in the efflux of cholesterol from the endocytic
circuits. Proteins, primarily Rab GTPases, which reduce endosomal cholesterol accumulation in
NPC1 cells would then be further characterized in their ability to facilitate intracellular
cholesterol transport.
The aim of the third part of the study was to characterize the potential function of Rab8 in
cellular cholesterol efflux from primary human macrophages. Macrophage foam cells are key
elements in the development of atherosclerotic lesions and prime targets for pharmacological
intervention. It is therefore crucial to understand the intracellular processes leading to ABCA1-
mediated cholesterol efflux. The goal of this part of the study was therefore to investigate the role
of Rab8 in the physiological setting of macrophage foam cell formation.
METHODS
Method Publication
Cell culture I, II, III
SDS-PAGE, Western blot, ECL (enhanced chemiluminescence) I, II, III
Northern blot I
Quantitative real-time PCR III
Surface biotinylation I, III
Adenovirus production and infection III
Semliki Forest virus production and infection II
RNA isolation I, II
Biochemical cholesterol measurements II, III
Biochemical cholesterol ester measurements II, III
Direct cholesterol efflux measurements II, III
GDI extraction of Rab proteins in vitro II
Cell fractionation II
Recombinant protein production and purification I
Polyclonal antibody production I
Immunocytochemistry I, II, III
Immunohistochemistry III
RNA interference II, III
Transient transfection I, II, III
Electroporation III
RESULTS AND DISCUSSION
1. Defective intracellular trafficking of NPC proteins in NPC disease (I)
1.1 Effects of individual mutations on the NPC1 protein Since the discovery of the two separate complementation groups in NPC it has been speculated
that the two NPC proteins act along the same pathway and might even interact. In order to assess
this, we sought to determine the subcellular localization of both NPC proteins, and analyze the
effects of disease causing mutations in NPC1 on the distribution of the NPC2 protein. We
identified three mutations, two of which were causative of NPC disease, C113R and a c-terminal
deletion mutant (delC), and a benign polymorphism P237S. Interestingly, the P237S substitution
has previously been regarded as a disease causing mutation (Kaminski et al., 2002). The P237S
substitution was present in ~5% of the alleles in the Finnish and Swedish population samples
studied. If P237S were to be a disease causing mutation one would expect the incidence of NPC1
disease to be much higher in the Finnish and Swedish populations. Moreover, overexpression of
the NPC1 protein harboring the P237S substitution fully restored cholesterol trafficking at the
cellular level. Although our results rule out P237S as a disease causing mutation it is still possible
that the mutation has a more subtle phenotype not leading to NPC1 disease.
In the case of the C-terminal deletion the case is clear-cut, since it leads to an unstable protein
which is rapidly degraded. The C113R substitution, however, results in a non-functional protein
that is partially mistargeted. We found the C113R protein to localize mainly to the ER, Rab7-
negative endosomes and the plasma membrane. Retarded export from the ER was confirmed by
metabolic labeling of the newly synthesized protein with 35S-methionine followed by
immunoprecipitation and endoglycosidaseH (endoH) digestion. The C113R protein showed a
decrease in the EndoH resistant fraction indicating that a smaller fraction of NPC1-C113R
reached the Golgi compared to the WT protein. What might be the mechanism for the decreased
export of NPC1-C113R from the ER? The C113R substitution resides in a highly conserved
region of the 240 amino acid (aa) luminal N-terminus containing 13 cysteine residues. Amino
MATTS LINDER
34
acids 55 through 165 encompass the “NPC” domain, a 112 aa stretch with high conservation
between NPC1 orthologues (Watari et al., 1999a). There are 8 conserved cysteine residues within
this domain, and site directed mutagenesis of four of these cysteines resulted in an inactive
protein that was localized to the limiting membrane of cholesterol-laden lysosomes in NPC cells
(Watari et al., 1999a). Presumably, the cysteine at aa position 113 participates in disulfide bridge
formation contributing to the tertiary structure of the N-terminal domain. This region was
recently shown to mediate oligomerisation of the NPC1 protein as well as to bind to cholesterol
and 25-hydroxycholesterol (Infante et al., 2008a). Interestingly the stochiometry of sterol binding
indicates that one cholesterol or hydroxycholesterol molecule binds to one dimer of the NPC1
protein. Whether or not dimerization or oligomerization of the NPC1 protein is a requirement for
sterol binding has not been assessed. However, it does not seem that sterols are required for the
dimerization of the protein since an N-terminal mutant defective in sterol binding (NPC1-Q79A)
showed the same pattern of oligomerisation as the WT protein (Infante et al., 2008a) (Linder and
Ikonen, unpublished observations). Also the NPC1-C113R protein forms oligomers indicating the
presumable changes in the n-terminal conformation are not sufficient to inhibit oligomerisation of
the protein (Linder and Ikonen, unpublished observations).
Although the C113R mutation clearly causes a defect in maturation of the protein it is curious
that the protein is also mistargeted after leaving the Golgi. The putative dileucine endosomal
targeting motif resides in the C-terminal of the protein, and ablation of this motif causes the
retention and degradation of the protein in the ER (Watari et al., 1999a). One explanation could
be that the C113R substitution interferes with binding to accessory proteins required for the
proper targeting of the protein. Alternatively, sterol binding at the N-terminal is required for the
proper localization and function of the protein. As stated above, the Q79A mutation causes a
defect in sterol binding, whereby 25-HC binding is abolished but residual cholesterol binding
capacity is retained. As for the C113R substitution, the cholesterol binding capacity has not been
assessed.
RESULTS AND DISCUSSION
35
1.2 Intracellular localization and regulation of the NPC2 protein in NPC1 cells
Characterization of the NPC2 protein has been hampered by the lack of high quality commercial
antibodies against NPC2. We generated polyclonal antibodies using recombinantly expressed
NPC2 fused to GST (amino acids 20-151, full length NPC2 w/o signal sequence) as an epitope.
The specificity of the antibody was confirmed by preincubation of the antibody with purified
recombinant protein prior to IF staining. Also the specificity has been confirmed by Western blot
using mouse NPC2 -/- samples (Mutka et al., 2004). In normal human fibroblasts we found the
endogenous NPC2 to have a punctate staining pattern. Double immunofluorescence staining
indicated that the majority of NPC2 was present in Lamp1-positive lysosomes. On the other
hand, in some cells, NPC2 was predominantly colocalized with gamma-adaptin, a marker for the
trans-Golgi network (TGN). Taken together, these results reconcile well with MPR regulated
shuttling of the NPC2 protein between the Golgi and endo/lysosomes. In contrast, NPC1 cells
showed an exclusively lysosomal localization of the NPC2 protein, indicating that there is a
sequesteration of the NPC2 protein in lysosomes. Consistent with this we saw a roughly 1.5-fold
increase in NPC2 protein by Western blotting. This might be a result of decreased degradation of
NPC2 or a compensatory upregulation of NPC2 gene transcription. Northern blot analysis
showed that the NPC2 transcript was significantly upregulated indicating that the increase in
NPC2 protein levels is due to a regulatory increase in NPC2 synthesis.
In the NPC1 fibroblasts the NPC2 protein was sequestered in the cholesterol-laden lysosomes
and very little Golgi like staining could be seen. In accordance with this there was also an
apparent redistribution of MPR to Lamp-1 positive organelles from the normally predominant
TGN localization. Transport of MPRs from late endosomes to the trans-Golgi is mediated by
Rab9 (Lombardi et al., 1993; Riederer et al., 1994) and the Rab9 effector TIP47 (Carroll et al.,
2001; Diaz and Pfeffer, 1998). Interestingly Rab9 function is perturbed in NPC1 fibroblasts
resulting in the accumulation of inactive Rab9 in late endosomes and lysosomal missorting and
degradation of MPR (Ganley and Pfeffer, 2006). It is therefore possible that NPC2 accumulates
as a result of defective Rab9 mediated transport steps. Whether or not this would have relevance
for the disease phenotype is unclear, since the putative site of NPC2 function is in the
MATTS LINDER
36
endo/lysosomal system, where NPC2 was shown to accumulate. In light of the increased NPC2
mRNA levels in NPC1 fibroblasts it would seem more likely that NPC2 is upregulated by a
compensatory mechanism as a result of the endosomal cholesterol accumulation. Alternatively,
transcription of NPC2 might fail to be downregulated due to the relative sterol deficiency in the
ER. It is therefore possible that the NPC proteins are transcriptionally regulated in concert to
maintain cholesterol efflux from the endo/lysosomal compartments, but an increase in one is not
sufficient to compensate for the loss of the other.
2. Complementation of Niemann-Pick type C disease by Rab overexpression (II)
Due to the diverse cellular pathology and the abundance of metabolites that accumulate in NPC
disease, it seems safe to argue that NPC is to a large extent a disorder of perturbed intracellular
membrane trafficking. Whatever the functions of the NPC proteins are and the nature of the
primary substrates, there is a gross stagnation of endosomal motility in NPC disease (Ko et al.,
2001; Zhang et al., 2001a). As of yet it has been difficult to pinpoint the exact nature of the
cellular lesion. It seems likely that the NPC2 protein is a bona fide cholesterol transporter,
arguing that in NPC2 disease cholesterol accumulation causes a malfunction in the endosomal
compartments. The case for NPC1 is less clear-cut. NPC1 has been proposed to function as a
transmembrane pump or transporter of yet unidentified substrates (Davies et al., 2000a), or as a
cholesterol transporter in tandem with NPC2 (Infante et al., 2008b).
Inactivation of the NPC1 proteins leads to the stagnation of late endosomal tubulovesicular
trafficking (Ko et al., 2001; Zhang et al., 2001a). This phenotype might be a result of impaired
function in select Rab GTPases, possibly through altered biophysical properties of the
membranes. Global inactivation of Rab GTPases with Rab-GDI causes retarded endosomal
cholesterol clearance in cultured human skin fibroblasts loaded with LDL (Höltta-Vuori et al.,
2000). Rab inhibition also disrupts complementation of NPC1 fibroblasts with overexpressed
NPC1 protein, indicating that this process is dependent on Rab proteins (Höltta-Vuori et al.,
2000). It was subsequently shown that overexpression of the late endosomal Rabs Rab7 and Rab9
was also able to complement the NPC1 phenotype. Of the recycling Rabs, only Rab4 has
previously been shown to complement the NPC1 phenotype whereas Rab11 does not. We
RESULTS AND DISCUSSION
37
therefore reasoned that overexpression of Rab proteins in NPC fibroblasts would be suitable for
identifying novel pathways of cholesterol transport from the endosomal system.
2.1 Use of filipin staining as a measure of cholesterol efflux from endosomal compartments
The fluorescent antibiotic filipin has been used extensively to visualize free cholesterol in fixed
cells. Previous complementation studies on NPC cells have used filipin staining as a measure of
cholesterol clearance from cells. In the study we used two NPC1 cell lines and one control cell
line. The GM3123 (I1061T/P237S) cell line from ATCC has been used extensively by other
laboratiories for complementation and was therefore chosen. The F92-116 NPC1 and F92-99
control cell line have been previously characterized in (I). It was immediately apparent by filipin
staining that the cholesterol accumulation phenotype varies heavily between the GM3123 and
F92-116 cell lines, with much more prominent perinuclear accumulation in the F92-116 line. The
heterogeneity of filipin staining in the GM3123 line was also striking. This is noteworthy since
previous complementation studies using single cell analysis have utilized this cell line.
We initially determined if filipin could be used as a quantitative measure of cholesterol clearance
from endosomes in NPC cells. Since the stochiometry of filipin binding to cholesterol has not
been determined we measured the correlation between filipin intensity and cellular free
cholesterol amounts. We found that there is good correlation between cellular free cholesterol
amounts and filipin intensity, establishing that a decrease in filipin intensity should be a good
measure of complementation. Indeed overexpression of Rab4-GFP and Rab7-GFP, which have
been previously shown to complement the NPC phenotype, as well as Rab8-GFP decreased
whole cell filipin staining. Whole cell filipin intensity can only be used as a measure of loss of
free cholesterol i.e. through efflux or esterification, not for redistribution of free cholesterol
within the cell. Also, a decrease in whole cell filipin staining might also reflect cholesterol efflux
from other compartments than endosomes, e.g. the plasma membrane. Whole cell filipin intensity
is therefore not an optimal measure of moblilization of free cholesterol from the endosomal
system. To more precisely measure mobilization of free cholesterol from the storage
endo/lysosomes we stained the cells with anti-Lamp1 antibodies and used this as a marker for the
storage organelles. After image acquisition, the area under the Lamp1 positive organelles was
MATTS LINDER
38
determined by image analysis and scored for filipin intensity, after subtraction of the PM filipin
fluorescence. By specifically scoring the endosomal cholesterol content the difference between
control F92-99 and NPC GM3123 fibroblasts becomes more apparent. Using this setup we
showed that overexpression of GFP-tagged Rab4, Rab7, and Rab8 specifically reduced the
cholesterol content of endosomes in NPC cells.
2.2 Rab8 restores sphingolipid trafficking in NPC fibroblasts Previous studies have established the restoration of glycosphingolipid transport in parallel with
filipin clearance during Rab overexpression (Choudhury et al., 2002; Choudhury et al., 2004). To
test whether Rab8 also restored glycosphingolipid trafficking in NPC cells we studied the
distribution of the cholera toxin subunit B (CTxB). CTxB binds to GM1 ganglioside and is
normally transported to the Golgi upon internalization but is retained in the endocytic
compartment in NPC cells (Choudhury et al., 2002; Sugimoto et al., 2001). Fluorescently labeled
CTxB localized to punctuate cytosolic structures in GM3123 fibroblasts but in Rab8-GFP
overexpressing cells CTxB colocalized with the Golgi marker GM130, indicating that Rab8
restored proper glycosphingolipid trafficking in NPC1 cells.
2.3 Rab8 is resistant to membrane retention during cholesterol loading Membrane cholesterol loading, as seen in NPC disease, disrupts Rab function by interfering with
Rab membrane extraction by Rab-GDI (Choudhury et al., 2004; Ganley and Pfeffer, 2006;
Lebrand et al., 2002). In NPC cells, and upon acLDL loading of mouse peritoneal macrophages,
we observed an upregulation of the Rab8 protein. To investigate whether this increase in Rab8
was caused by sequestration of Rab8 in cholesterol enriched membranes, we incubated cellular
membranes with Rab-GDI. Incubation with 2µM purified GDI solubilized roughly 70% of
membrane bound Rab8 independent of cholesterol load. In contrast, Rab7 solubility was reduced
in NPC cells in accordance with previous reports (Lebrand et al., 2002). Apparently the
membranes that Rab8 resides in do not accumulate cholesterol in NPC cells, and therefore the
membrane extractability remains unaltered. Interestingly, the overall membrane extractability of
RESULTS AND DISCUSSION
39
Rab8 seemed to be greater for Rab8 compared to Rab7. Rab8 is only geranylgeranylated at a
single site as opposed to the double prenylation of most other Rab proteins e.g. Rab7, which
might confer resistance to membrane sequestration during cholesterol loading. Also, the affinity
for Rab-GDI depends on the length of the hypervariable domain of the Rab protein. The high
membrane extractability of Rab8 might therefore help Rab8 to stay functional during cholesterol
enrichment of membranes.
2.4 Rab8 siRNA induces a cholesterol accumulation phenotype So far only global inactivation of Rab proteins with Rab-GDI has been shown to induce a
cholesterol storage phenotype, whereas RNAi of specific Rabs has been ineffective. Ganley and
coworkers showed that specific knock-down of Rab9 did not induce endosomal cholesterol
accumulation (Ganley and Pfeffer, 2006). It is therefore possible that overexpression of certain
Rab proteins stimulates cholesterol transport in redundant or marginal pathways. To evaluate if
Rab8 function is necessary for cholesterol trafficking in normal cells we knocked down the Rab8
protein using RNA interference. Transfection of primary human fibroblasts with Rab8 shRNA
resulted in an increase in intracellular cholesterol, as assessed by filipin staining, that partly
localized to Lamp1 positive organelles. The cholesterol accumulation was also accompanied by a
cholesterol efflux defect to apoA-I. Also, Rab8 RNAi resulted in decreased cholesterol
esterification, indicating that the mobilized cholesterol was not targeted to the ER.
MATTS LINDER
40
3. Rab8 regulates cholesterol processing in primary human macrophages (III)
3.1 Rab8 is expressed in macrophages present in atherosclerotic lesions in vivo
To assess if Rab8 might be beneficial in foam cell reduction we opted to work on primary human
macrophages. Initially, we conducted immunohistochemical staining of human coronary artery
sections of atherosclerotic lesions. Using a polyclonal antibody against Rab8 we found Rab8 to
be expressed in cells surrounding the necrotic core of the lesion. To confirm that these cells were
macrophages we double stained the sections with anti-Rab8 and anti-CD68 antibodies. The Rab8
expressing cells showed clear co-localization with CD68 indicating that the cells were indeed
macrophages. The results indicate that Rab8 might have a physiological role in the setting of
macrophage foam cell formation, but whether this is directly related to cholesterol metabolism
can not be determined from these experiments.
3.2 Adenoviral overexpression of Rab8 in vitro increases ABCA1 dependent cholesterol efflux to apoA-I
We used an adenoviral expression system to overexpress Rab8 in primary human macrophages.
Infection of macrophages with Rab8 induced marked morphological changes, as has previously
been shown in other cell types. The macrophages displayed multiple cell protrusions positive for
Rab8. To assess if these cells also showed an increased capacity to withstand foam cell formation
we incubated control (GFP) or Rab8-myc infected macrophages in the presence of acLDL and
apoA-I. Cells infected with Rab8 accumulated less total cholesterol as compared to the control
cells. Interestingly this decrease in total cholesterol could be accounted for by the total decrease
in cholesteryl esters indicating that the cholesteryl ester pool is mainly affected. This could be
due rapid recycling of cholesterol from the endosomes to the cell surface, thereby decreasing the
cholesterol pool that is available for re-esterification in the ER. Alternatively, Rab8 might
facilitate the mobilization of cholesterol from lipid droplets. Since the amount of free cholesterol
remained constant during Rab8 overexpression it would seem that the mobilized cholesterol is
readily effluxed from the cells, suggesting involvement of the ABCA1 transporter. In accordance
with this we saw a striking, 2.5-fold increase in the amount of ABCA protein in the Rab8
RESULTS AND DISCUSSION
41
overexpressing cells. This increase of ABCA1 was unlikely to be caused by an increase in
ABCA1 transcription through e.g. LXR activation since we saw no significant upregulation of
the ABCA1 mRNA in quantitative real-time PCR. It is therefore logical to assume that Rab8
induces the stabilization of ABCA1 on the cell surface, and thereby retards the degradation of the
protein. Stabilization might occur through increased exposition of ABCA1 to extracellular apoA-
I through enhanced trafficking of ABCA1 to the PM. It has previously been shown that
degradation of ABCA1 is regulated by internalization of the protein, and that apoA-I decreases
internalization by stabilizing ABCA1 at the cell surface (Wang et al., 2003). Internalization of
ABCA1 is dependent on the C-terminal PEST sequence (Chen et al., 2005). Given the
established effects of Rab8 on actin remodelling it is also feasible that ABCA1 stabilization is an
actin-mediated effect. ABCA1 has been shown to interact with members of the syntrophin family
of proteins ( 1, 1 and 2) (Buechler et al., 2002; Munehira et al., 2004; Okuhira et al., 2005).
Overexpression of 1- syntrophin increases the half-life of ABCA1 and facilitates cholesterol
efflux (Munehira et al., 2004). Co-expression of 1-syntrophin and ABCA1 induces the
formation of ABCA1 clusters at the PM, stabilizing the ABCA1 protein and increasing efflux
capacity (Okuhira et al., 2005). The syntrophins link ABCA1 to the actin cytoskeleton by
forming a complex with the scaffolding protein utrophin. Rab8 might therefore facilitate
cholesterol efflux by providing the actin scaffold for stabilization of the
ABCA1/syntrophin/utrophin complex.
3.3 Rab8 RNAi inhibits cholesterol efflux and regulates ABCA1 trafficking in primary human macrophages
To establish if Rab8 is essential for cholesterol transport in primary human macrophages we used
RNA silencing to knock down the Rab8 protein. We previously showed that Rab8 siRNA in
primary human macrophages causes retention of cholesterol within the cells as assessed by filipin
staining (II). In the primary human macrophages we opted to biochemically assess the cholesterol
amounts in Rab8 knock-down and control cells. Two days after electroporation of the siRNA the
macrophages were loaded with 50 g/ml acLDL. This resulted in a marked, 3-fold increase in
total cellular cholesterol and an increase of the cholesteryl ester fraction from 3.5% to 45%.
MATTS LINDER
42
After acLDL loading the cells were incubated with 10 g/ml apoA-I for 18h. In control cells, the
total cellular cholesterol levels decreased by 40% during the incubation period, whereas the Rab8
depleted cells failed to efflux significant amounts of cholesterol. To confirm that the substrate
pool for efflux was derived from the endocytosed lipoprotein particles we labeled acLDL with 3H cholesteryl oleate. Scintillation counting of the cells after loading confirmed equal uptake of
the label. However after incubation with apoA-I, the Rab8 depleted cells failed to efflux the 3H cholesterol, in accordance with the previous results.
After endocytosis the cholesteroyl esters of the LDL particle are hydrolysed by lysosomal acid
lipase (LAL), and it is conceivable that Rab8 siRNA interferes with this step and therefore
inhibits cholesterol mobilization from the cells. This is, however, an unlikely scenario since Rab8
shRNA causes cholesterol accumulation in primary human fibroblasts as assessed by filipin
staining, and filipin only stains free cholesterol. There are a number of possibilities how Rab8
siRNA might interfere with cholesterol efflux. Firstly, Rab8 depletion might inhibit the
trafficking of recycling endosomes to the cell surface. In accordance, filipin staining of primary
human macrophages showed cholesterol accumulation in both Lamp1-positive and Lamp1-
negative organelles. This accumulation is separate from that seen in NPC1 disease, which is
primarily lysosomal in nature, indicating that Rab8 regulates a down-stream event in cholesterol
trafficking. As discussed above, one possibility is that Rab8 controls the stabilization of ABCA1
on the PM. To assess the effect of Rab8 on the plasma membrane localization of ABCA1 we
used RNA silencing to knock down Rab8, followed by surface biotinylation of ABCA1. In Rab8
siRNA treated cells there was a significant, 30% decrease in the fraction of ABCA1 on the
plasma membrane, indicating that Rab8 regulates the transport of ABCA1 to the PM or
stabilization of the protein on the PM.
It is possible that Rab8 controls the trafficking or recycling of ABCA1 to the cell surface. Due to
a lack of good antibodies the precise cellular localization of ABCA1 has been hard to pinpoint,
although it is clear through surface biotinylation experiments that a large portion of the protein
resides at the plasma membrane. By overexpressing GFP-tagged ABCA1 it has also been
suggested that ABCA1 resides in endosomal compartments. It seems that a part of this pool of
ABCA1 is destined for lysosomal degradation, whereas a part is recycled back to the PM
RESULTS AND DISCUSSION
43
(Neufeld et al., 2001). Yet, the specific recycling route has not been identified. Since Rab8
affects the cell surface distribution of ABCA1 we studied whether Rab8 siRNA would also have
an effect on the intracellular distribution of ABCA1-GFP. Previous studies on ABCA1-GFP have
mostly been carried out in HeLa cells. In a study by Neufeld et al, ABCA-GFP was localized to
the plasma membrane and Lamp2-positive vesicles, but not to TfR-positive vesicles (Neufeld et
al., 2001). We also found ABCA1-GFP to localize to the PM in HeLa cells, mainly to cellular
lamellipodia like protrusions and to intracellular tubules, both colocalizing with 1 integrin. Rab8
siRNA caused a decrease in cellular protrusions in the ABCA1 expressing cells and retention of
ABCA1 and 1 integrin in intracellular tubular compartments, indicating that the trafficking of
b1 integrin and ABCA1-GFP are interconnected. In accordance with previous results (Neufeld et
al., 2001) we did not find ABCA1 to colocalize with TfR. However, in Rab8 siRNA treated cells
ABCA1 accumulated perinuclearily, in a partially TfR positive structure. Taken together, the
results indicate that Rab8 also regulates the intracellular trafficking of ABCA1.
CONCLUSIONS AND FUTURE PROSPECTS
In this study (I) we have identified a mutation in the NPC1 protein that disrupts the intracellular
trafficking of the protein, leading to a severe infantile form of NPC disease. The study
demonstrates the importance of the correct localization of NPC1 within the cell. Also, the
regulation of NPC2 gene transcription seems to be dependent on proper NPC1 function
indicating that these proteins might be functionally linked. Of the various domains in the NPC1
protein, the SSD is of outstanding interest. There is high sequence homology within this domain
between SCAP, HMGR, NPC1L1 and NPC1 (Kuwabara and Labouesse, 2002). Therefore, a
crucial challenge will be to establish whether the SSD performs a similar sterol-sensing function
in NPC1 as has been described for SCAP and HMGR, and if so, to establish the sterols that
regulate NPC1 function.
The lessons learned from NPC1 might also have impact on our understanding of the regulation of
other SSD containing proteins, e.g. NPC1L1. Conversely, mutational and functional analysis of
the SSD within NPC1L1 might shed light on the function of this domain within NPC1. It has
already been established that sterols regulate the intracellular trafficking of NPC1L1. Upon sterol
depletion NPC1L1 translocates from the endocytic recycling compartment to the plasma
membrane where it facilitates cholesterol uptake. It would be interesting to study whether the
trafficking of NPC1L1 is dependent on an intact SSD. A recent study also showed that
cholesterol induces the internalization of NPC1L1 in intestinal cells, and that the cholesterol
lowering drug ezetimibe blocks this internalization (Ge et al., 2008). It would therefore be of
interest to determine if ezetimibe added to cells in vitro could block NPC1 mediated
tubulovesicular trafficking. If so, this could suggest that the function of NPC1 could be the
generation of vesicle budding sites as proposed by Yiannis Ioannou (Ioannou, 2005). In this
model NPC1 would function by flipping membrane lipids such as fatty acids or sphingosine,
thereby facilitating an outward bending membrane curvature. However, any theory on the
function of NPC1 should also take in to account the cholesterol binding properties of the protein.
In analogy to NPC1L1 internalization, cholesterol might also serve as an activating trigger for
NPC1 function. A regulatory role for cholestrerol binding could explain why an NPC1 N-
terminal mutant with lack of 25-hydroxycholesterol binding and severely attenuated cholesterol
CONCLUSIONS AND FUTURE PROSPECTS
45
binding had no apparent cellular phenotype (Infante et al., 2008a). It is feasible that the residual
cholesterol binding capacity is sufficient to serve a regulatory function. The bulk cholesterol
transport would then be mediated by NPC2 to specific membrane domains created by NPC1.
NPC2 is capable of transferring cholesterol from the N-terminal domain of NPC1 to acceptor
membranes (Infante et al., 2008b). It is also possible that NPC2 could work in the opposite way,
transferring cholesterol from inner membranes to NPC1 at the limiting membrane. In this model
cholesterol transfer from inner membranes could be facilitated by LBPA/BMP, which has been
shown to increase the sterol transfer capacity of NPC2 when present in the donor membrane
(Cheruku et al., 2006).
To date, most studies aimed at elucidating the function of NPC1 have focused on transient
transfection to revert the accumulation phenotype. An alternative approach could be the acute
knock-down of NPC1 or NPC2, to assess which metabolite(s) accumulate first. This might help
to resolve the long standing hen-and-egg issue that has puzzled NPC researchers, i.e. which
comes first, cholesterol or sphingolipid accumulation. However, such a study should also take in
to account the sterol status of the cell, as well as the inherent difference in the sensitivities
between assays for cholesterol and sphingolipid accumulation.
Our studies (II, III) are the first to identify a specific Rab-GTPase localized outside the
endosomal system that affects the recycling of cholesterol from the endosomes to the PM. We
have also shown that Rab8 is involved in the cellular trafficking of ABCA1. It is clear that the
processes governing ABCA1-mediated cholesterol efflux to apoA-I are highly complex,
involving intracellular signaling cascades and cytoskeleton reorganization. Given the role of
Rab8 in inducing cell polarity and actin as well as microtubule reorganization, a likely scenario is
that Rab8 promotes cholesterol efflux by creating platforms for cholesterol efflux at the leading
edge of the cell. Although the contribution of endocytosed ABCA1/apoA-1 to cellular cholesterol
efflux remains to be resolved, it is quite clear that ABCA1 is regulated post-transcriptionally
through endocytosis and recycling of the protein. According to our results Rab8 has a role in this
process, but the precise mechanism of Rab8 function remains to be established. We have shown
that knock-down of Rab8 causes the retention of ABCA1 within the cell in 1 integrin and TfR
receptor positive structures. It remains to be established whether ABCA1 containing vesicles
MATTS LINDER
46
utilize Rab8 directly, e.g. for recruitment of myosin motors, or if the effect is secondary e.g.
through altered actin or microtubule organization.
Our results also suggest that Rab8 recycles cholesterol from the endosomal system back to the
plasma membrane. The next challenge will therefore be to identify the precise recycling
pathway(s) regulated by Rab8 other Rab proteins involved in this process. The strategy adopted
in this study i.e. transfection of select Rab proteins and single cell assessment of filipin intensity
is quite labor intensive. In order to identify the Rab proteins involved in intracellular cholesterol
trafficking there is therefore a need for high throughput approaches, for instance, utilization of
siRNA libraries with automated analysis. Alternatively, a Rab-GAP library such as the one used
to identify Rab-GTPases involved in primary cilium formation could be used (Yoshimura et al.,
2007). The challenge of these approaches is, however, to find a clear read-out for cholesterol
trafficking suitable for automated analysis, preferably in living cells. The filipin staining process
is not suitable for high throughput applications. To this end one might utilize fluorescent
cholesterol analogs to measure changes in uptake, intracellular distribution and efflux. One
candidate probe would be BODIPY-cholesterol which has been shown to behave similarly to
cholesterol in normal and storage-disease cells (Höltta-Vuori et al., 2008).
Finally, it would be interesting to carry out in vivo studies in mice. The Rab8-/- mice described
by Sato and colleagues could be used for such studies, for instance to assess the serum
lipoprotein particle composition during low and high cholesterol diet (Sato et al., 2007).
Alternatively, generation of transgenic mice between Rab8-/- and LDLr-/- (or apoE-/-) mice
might be used to assess the effect of Rab8 deficiency on atherosclerotic plaque development.
Also, bone marrow transplants from Rab8 KO mice to LDLr -/- mice might be used to directly
assess the contribution of Rab8 in foam cell development.
Macrophage RCT measurements in vivo could also be carried out by adenoviral infection of
macrophages with Rab8, and metabolic labeling with [3H]-cholesterol in vitro, followed by
intraperitoneal injection of infected macrophages. RCT could then be measured by the
appearance of the [3H]-tracer in the plasma, liver and feces (Tanigawa et al., 2007). Together
CONCLUSIONS AND FUTURE PROSPECTS
47
these approaches should determine the overall contribution of Rab8 in macrophage RCT and
atherosclerotic lesion development.
ACKNOWLEDGEMENTS
I would like to take this opportunity to thank the numerous people that have contributed to the
success of this work.
Special thanks go out to my supervisor, Professor Elina Ikonen, for her optimism and
encouragement.
This work would, of course, not have been possible without the generous help of my colleagues
within and outside the laboratory. I would like to thank all the members of the Ikonen Lab for
their friendship and support. Anna Uro is warmly thanked for her help. Likewise, I would like to
thank Titta Blom, Maarit Hölttä-Vuori, Vilja Pietiäinen, Aino-Liisa Mutka and Riikka-Liisa
Uronen for their contributions. I also wish to thank Seija Puomilahti, Liisa Arala and Birgitta
Rantala for their much needed assistance. Additionally, I would like to thank Docent Johan
Peränen for so generously sharing constructs and antibodies. Professor Ismo Virtanen, Professor
Christian Ehnholm, Adjunct Professor Vesa Olkkonen and Adjunct Professor Matti Jauhiainen
have also been very generous in sharing materials and know-how.
I would like to thank the former and current heads of the departments at which the work has been
conducted. Professors Christian Ehnholm and Leena Palotie at the National Institute for Health
and Welfare (formerly National Public Health Institute); Professor Mart Saarma, director of the
Institute of Biotechnology, University of Helsinki. Also, Professor Ismo Virtanen and Professor
Esa Korpi of the Institute of Biomedicine, University of Helsinki, are acknowledged for
providing excellent facilities and working environments.
I would also like to thank the members of my thesis committee group, Docent Ismo Ulmanen and
Docent Mikko Frilander, for their advice.
Docent Jussi Jäntti and Professor Markku Savolainen are warmly thanked for reviewing the
thesis.
49
I also wish to acknowledge the continuing support of my friends, with special thanks to Ron
Liebkind for helpful discussions.
I am very grateful to my family, especially my parents Birgitta and Ewert for their continuous
support, as well as my sister Nina for all her help.
Carina and my son Cedric are warmly thanked for their support and understanding.
Financial support from Finska Läkaresällskapet, the Finnish Foundation for Cardiovascular
Research, Oskar Öflunds Stiftelse, Medicinka understödsföreningen Liv och Hälsa and Helsinki
Biomedical Graduate School is gratefully acknowledged.
REFERENCES Alder-Baerens, N., P. Muller, A. Pohl, T. Korte, Y. Hamon, G. Chimini, T. Pomorski, and A.
Herrmann. 2005. Headgroup-specific exposure of phospholipids in ABCA1-expressing cells. J Biol Chem. 280:26321-9.
Alory, C., and W.E. Balch. 2001. Organization of the Rab-GDI/CHM superfamily: the functional basis for choroideremia disease. Traffic. 2:532-43.
Andres, D.A., M.C. Seabra, M.S. Brown, S.A. Armstrong, T.E. Smeland, F.P. Cremers, and J.L. Goldstein. 1993. cDNA cloning of component A of Rab geranylgeranyl transferase and demonstration of its role as a Rab escort protein. Cell. 73:1091-9.
Ang, A.L., H. Folsch, U.M. Koivisto, M. Pypaert, and I. Mellman. 2003. The Rab8 GTPase selectively regulates AP-1B-dependent basolateral transport in polarized Madin-Darby canine kidney cells. J Cell Biol. 163:339-50.
Ang, A.L., T. Taguchi, S. Francis, H. Folsch, L.J. Murrells, M. Pypaert, G. Warren, and I. Mellman. 2004. Recycling endosomes can serve as intermediates during transport from the Golgi to the plasma membrane of MDCK cells. J Cell Biol. 167:531-43.
Armstrong, J., N. Thompson, J.H. Squire, J. Smith, B. Hayes, and R. Solari. 1996. Identification of a novel member of the Rab8 family from the rat basophilic leukaemia cell line, RBL.2H3. J Cell Sci. 109 (Pt 6):1265-74.
Au, J.S., C. Puri, G. Ihrke, J. Kendrick-Jones, and F. Buss. 2007. Myosin VI is required for sorting of AP-1B-dependent cargo to the basolateral domain in polarized MDCK cells. J Cell Biol. 177:103-14.
Babalola, J.O., M. Wendeler, B. Breiden, C. Arenz, G. Schwarzmann, S. Locatelli-Hoops, and K. Sandhoff. 2007. Development of an assay for the intermembrane transfer of cholesterol by Niemann-Pick C2 protein. Biol Chem. 388:617-26.
Blanchette-Mackie, E.J., N.K. Dwyer, L.M. Amende, H.S. Kruth, J.D. Butler, J. Sokol, M.E. Comly, M.T. Vanier, J.T. August, R.O. Brady, and et al. 1988. Type-C Niemann-Pick disease: low density lipoprotein uptake is associated with premature cholesterol accumulation in the Golgi complex and excessive cholesterol storage in lysosomes. Proc Natl Acad Sci U S A. 85:8022-6.
Bodzioch, M., E. Orso, J. Klucken, T. Langmann, A. Bottcher, W. Diederich, W. Drobnik, S. Barlage, C. Buchler, M. Porsch-Ozcurumez, W.E. Kaminski, H.W. Hahmann, K. Oette, G. Rothe, C. Aslanidis, K.J. Lackner, and G. Schmitz. 1999. The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease. Nat Genet. 22:347-51.
Brooks-Wilson, A., M. Marcil, S.M. Clee, L.H. Zhang, K. Roomp, M. van Dam, L. Yu, C. Brewer, J.A. Collins, H.O. Molhuizen, O. Loubser, B.F. Ouelette, K. Fichter, K.J. Ashbourne-Excoffon, C.W. Sensen, S. Scherer, S. Mott, M. Denis, D. Martindale, J. Frohlich, K. Morgan, B. Koop, S. Pimstone, J.J. Kastelein, J. Genest, Jr., and M.R. Hayden. 1999. Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency. Nat Genet. 22:336-45.
Bucci, C., P. Thomsen, P. Nicoziani, J. McCarthy, and B. van Deurs. 2000. Rab7: a key to lysosome biogenesis. Mol Biol Cell. 11:467-80.
REFERENCES
51
Buechler, C., A. Boettcher, S.M. Bared, M.C. Probst, and G. Schmitz. 2002. The carboxyterminus of the ATP-binding cassette transporter A1 interacts with a beta2-syntrophin/utrophin complex. Biochem Biophys Res Commun. 293:759-65.
Carroll, K.S., J. Hanna, I. Simon, J. Krise, P. Barbero, and S.R. Pfeffer. 2001. Role of Rab9 GTPase in facilitating receptor recruitment by TIP47. Science. 292:1373-6.
Carstea, E.D., J.A. Morris, K.G. Coleman, S.K. Loftus, D. Zhang, C. Cummings, J. Gu, M.A. Rosenfeld, W.J. Pavan, D.B. Krizman, J. Nagle, M.H. Polymeropoulos, S.L. Sturley, Y.A. Ioannou, M.E. Higgins, M. Comly, A. Cooney, A. Brown, C.R. Kaneski, E.J. Blanchette-Mackie, N.K. Dwyer, E.B. Neufeld, T.Y. Chang, L. Liscum, J.F. Strauss, 3rd, K. Ohno, M. Zeigler, R. Carmi, J. Sokol, D. Markie, R.R. O'Neill, O.P. van Diggelen, M. Elleder, M.C. Patterson, R.O. Brady, M.T. Vanier, P.G. Pentchev, and D.A. Tagle. 1997. Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science. 277:228-31.
Casey, P.J., and M.C. Seabra. 1996. Protein prenyltransferases. J Biol Chem. 271:5289-92. Cavelier, C., I. Lorenzi, L. Rohrer, and A. von Eckardstein. 2006. Lipid efflux by the ATP-
binding cassette transporters ABCA1 and ABCG1. Biochim Biophys Acta. 1761:655-66. Chabrillat, M.L., C. Wilhelm, C. Wasmeier, E.V. Sviderskaya, D. Louvard, and E. Coudrier.
2005. Rab8 regulates the actin-based movement of melanosomes. Mol Biol Cell. 16:1640-50.
Chang, T.Y., C.C. Chang, S. Lin, C. Yu, B.L. Li, and A. Miyazaki. 2001. Roles of acyl-coenzyme A:cholesterol acyltransferase-1 and -2. Curr Opin Lipidol. 12:289-96.
Chavrier, P., J.P. Gorvel, E. Stelzer, K. Simons, J. Gruenberg, and M. Zerial. 1991. Hypervariable C-terminal domain of rab proteins acts as a targeting signal. Nature. 353:769-72.
Chen, W., N. Wang, and A.R. Tall. 2005. A PEST deletion mutant of ABCA1 shows impaired internalization and defective cholesterol efflux from late endosomes. J Biol Chem. 280:29277-81.
Cheruku, S.R., Z. Xu, R. Dutia, P. Lobel, and J. Storch. 2006. Mechanism of cholesterol transfer from the Niemann-Pick type C2 protein to model membranes supports a role in lysosomal cholesterol transport. J Biol Chem. 281:31594-604.
Chikh, K., S. Vey, C. Simonot, M.T. Vanier, and G. Millat. 2004. Niemann-Pick type C disease: importance of N-glycosylation sites for function and cellular location of the NPC2 protein. Mol Genet Metab. 83:220-30.
Choudhury, A., M. Dominguez, V. Puri, D.K. Sharma, K. Narita, C.L. Wheatley, D.L. Marks, and R.E. Pagano. 2002. Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells. J Clin Invest. 109:1541-50.
Choudhury, A., D.K. Sharma, D.L. Marks, and R.E. Pagano. 2004. Elevated endosomal cholesterol levels in Niemann-Pick cells inhibit rab4 and perturb membrane recycling. Mol Biol Cell. 15:4500-11.
Chroni, A., T. Liu, M.L. Fitzgerald, M.W. Freeman, and V.I. Zannis. 2004. Cross-linking and lipid efflux properties of apoA-I mutants suggest direct association between apoA-I helices and ABCA1. Biochemistry. 43:2126-39.
Cuchel, M., and D.J. Rader. 2006. Macrophage reverse cholesterol transport: key to the regression of atherosclerosis? Circulation. 113:2548-55.
MATTS LINDER
52
Davies, J.P., F.W. Chen, and Y.A. Ioannou. 2000a. Transmembrane molecular pump activity of Niemann-Pick C1 protein. Science. 290:2295-8.
Davies, J.P., and Y.A. Ioannou. 2000. Topological analysis of Niemann-Pick C1 protein reveals that the membrane orientation of the putative sterol-sensing domain is identical to those of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding protein cleavage-activating protein. J Biol Chem. 275:24367-74.
Davies, J.P., B. Levy, and Y.A. Ioannou. 2000b. Evidence for a Niemann-pick C (NPC) gene family: identification and characterization of NPC1L1. Genomics. 65:137-45.
Del Toro, D., J. Alberch, F. Lazaro-Dieguez, R. Martin-Ibanez, X. Xifro, G. Egea, and J.M. Canals. 2009. Mutant Huntingtin Impairs Post-Golgi Trafficking to Lysosomes by Delocalizing Optineurin/Rab8 Complex from the Golgi Apparatus. Mol Biol Cell.
Denis, M., B. Haidar, M. Marcil, M. Bouvier, L. Krimbou, and J. Genest, Jr. 2004. Molecular and cellular physiology of apolipoprotein A-I lipidation by the ATP-binding cassette transporter A1 (ABCA1). J Biol Chem. 279:7384-94.
Denis, M., Y.D. Landry, and X. Zha. 2008. ATP-binding cassette A1-mediated lipidation of apolipoprotein A-I occurs at the plasma membrane and not in the endocytic compartments. J Biol Chem. 283:16178-86.
Diaz, E., and S.R. Pfeffer. 1998. TIP47: a cargo selection device for mannose 6-phosphate receptor trafficking. Cell. 93:433-43.
von Eckardstein, A., A. Chirazi, S. Schuler-Luttmann, M. Walter, J.J. Kastelein, J. Geisel, J.T. Real, R. Miccoli, G. Noseda, G. Hobbel, and G. Assmann. 1998. Plasma and fibroblasts of Tangier disease patients are disturbed in transferring phospholipids onto apolipoprotein A-I. J Lipid Res. 39:987-98.
Espenshade, P.J., and A.L. Hughes. 2007. Regulation of sterol synthesis in eukaryotes. Annu Rev Genet. 41:401-27.
Fitzgerald, M.L., A.L. Morris, A. Chroni, A.J. Mendez, V.I. Zannis, and M.W. Freeman. 2004. ABCA1 and amphipathic apolipoproteins form high-affinity molecular complexes required for cholesterol efflux. J Lipid Res. 45:287-94.
Fitzgerald, M.L., A.L. Morris, J.S. Rhee, L.P. Andersson, A.J. Mendez, and M.W. Freeman. 2002. Naturally occurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I. J Biol Chem. 277:33178-87.
Friedland, N., H.L. Liou, P. Lobel, and A.M. Stock. 2003. Structure of a cholesterol-binding protein deficient in Niemann-Pick type C2 disease. Proc Natl Acad Sci U S A. 100:2512-7.
Frolov, A., S.E. Zielinski, J.R. Crowley, N. Dudley-Rucker, J.E. Schaffer, and D.S. Ory. 2003. NPC1 and NPC2 regulate cellular cholesterol homeostasis through generation of low density lipoprotein cholesterol-derived oxysterols. J Biol Chem. 278:25517-25.
Ganley, I.G., and S.R. Pfeffer. 2006. Cholesterol accumulation sequesters Rab9 and disrupts late endosome function in NPC1-deficient cells. J Biol Chem. 281:17890-9.
Ge, L., J. Wang, W. Qi, H.H. Miao, J. Cao, Y.X. Qu, B.L. Li, and B.L. Song. 2008. The cholesterol absorption inhibitor ezetimibe acts by blocking the sterol-induced internalization of NPC1L1. Cell Metab. 7:508-19.
Gillotte, K.L., M. Zaiou, S. Lund-Katz, G.M. Anantharamaiah, P. Holvoet, A. Dhoest, M.N. Palgunachari, J.P. Segrest, K.H. Weisgraber, G.H. Rothblat, and M.C. Phillips. 1999. Apolipoprotein-mediated plasma membrane microsolubilization. Role of lipid affinity and
REFERENCES
53
membrane penetration in the efflux of cellular cholesterol and phospholipid. J Biol Chem. 274:2021-8.
Glass, C.K., and J.L. Witztum. 2001. Atherosclerosis. the road ahead. Cell. 104:503-16. Goldstein, J.L., R.A. DeBose-Boyd, and M.S. Brown. 2006. Protein sensors for membrane
sterols. Cell. 124:35-46. Hansson, G.K., and P. Libby. 2006. The immune response in atherosclerosis: a double-edged
sword. Nat Rev Immunol. 6:508-19. Hassan, H.H., D. Bailey, D.Y. Lee, I. Iatan, A. Hafiane, I. Ruel, L. Krimbou, and J. Genest. 2008.
Quantitative analysis of ABCA1-dependent compartmentalization and trafficking of apolipoprotein A-I: implications for determining cellular kinetics of nascent high density lipoprotein biogenesis. J Biol Chem. 283:11164-75.
Hassan, H.H., M. Denis, D.Y. Lee, I. Iatan, D. Nyholt, I. Ruel, L. Krimbou, and J. Genest. 2007. Identification of an ABCA1-dependent phospholipid-rich plasma membrane apolipoprotein A-I binding site for nascent HDL formation: implications for current models of HDL biogenesis. J Lipid Res. 48:2428-42.
Hattula, K., J. Furuhjelm, A. Arffman, and J. Peränen. 2002. A Rab8-specific GDP/GTP exchange factor is involved in actin remodeling and polarized membrane transport. Mol Biol Cell. 13:3268-80.
Hattula, K., and J. Peränen. 2000. FIP-2, a coiled-coil protein, links Huntingtin to Rab8 and modulates cellular morphogenesis. Curr Biol. 10:1603-6.
Higgins, M.E., J.P. Davies, F.W. Chen, and Y.A. Ioannou. 1999. Niemann-Pick C1 is a late endosome-resident protein that transiently associates with lysosomes and the trans-Golgi network. Mol Genet Metab. 68:1-13.
Höltta-Vuori, M., J. Maatta, O. Ullrich, E. Kuismanen, and E. Ikonen. 2000. Mobilization of late-endosomal cholesterol is inhibited by Rab guanine nucleotide dissociation inhibitor. Curr Biol. 10:95-8.
Höltta-Vuori, M., K. Tanhuanpaa, W. Möbius, P. Somerharju, and E. Ikonen. 2002. Modulation of cellular cholesterol transport and homeostasis by Rab11. Mol Biol Cell. 13:3107-22.
Höltta-Vuori, M., R.L. Uronen, J. Repakova, E. Salonen, I. Vattulainen, P. Panula, Z. Li, R. Bittman, and E. Ikonen. 2008. BODIPY-cholesterol: a new tool to visualize sterol trafficking in living cells and organisms. Traffic. 9:1839-49.
Huber, L.A., P. Dupree, and C.G. Dotti. 1995. A deficiency of the small GTPase rab8 inhibits membrane traffic in developing neurons. Mol Cell Biol. 15:918-24.
Huber, L.A., S. Pimplikar, R.G. Parton, H. Virta, M. Zerial, and K. Simons. 1993. Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane. J Cell Biol. 123:35-45.
Infante, R.E., A. Radhakrishnan, L. Abi-Mosleh, L.N. Kinch, M.L. Wang, N.V. Grishin, J.L. Goldstein, and M.S. Brown. 2008a. Purified NPC1 protein: II. Localization of sterol binding to a 240-amino acid soluble luminal loop. J Biol Chem. 283:1064-75.
Infante, R.E., M.L. Wang, A. Radhakrishnan, H.J. Kwon, M.S. Brown, and J.L. Goldstein. 2008b. NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes. Proc Natl Acad Sci U S A. 105:15287-92.
Ioannou, Y.A. 2005. Guilty until proven innocent: the case of NPC1 and cholesterol. Trends Biochem Sci. 30:498-505.
MATTS LINDER
54
Kaminski, W.E., H.H. Klunemann, B. Ibach, C. Aslanidis, H.E. Klein, and G. Schmitz. 2002. Identification of novel mutations in the NPC1 gene in German patients with Niemann-Pick C disease. J Inherit Metab Dis. 25:385-9.
Kaptzan, T., S.A. West, E.L. Holicky, C.L. Wheatley, D.L. Marks, T. Wang, K.B. Peake, J. Vance, S.U. Walkley, and R.E. Pagano. 2009. Development of a Rab9 transgenic mouse and its ability to increase the lifespan of a murine model of Niemann-Pick type C disease. Am J Pathol. 174:14-20.
Kirchhoff, C., C. Osterhoff, and L. Young. 1996. Molecular cloning and characterization of HE1, a major secretory protein of the human epididymis. Biol Reprod. 54:847-56.
Ko, D.C., J. Binkley, A. Sidow, and M.P. Scott. 2003. The integrity of a cholesterol-binding pocket in Niemann-Pick C2 protein is necessary to control lysosome cholesterol levels. Proc Natl Acad Sci U S A. 100:2518-25.
Ko, D.C., M.D. Gordon, J.Y. Jin, and M.P. Scott. 2001. Dynamic movements of organelles containing Niemann-Pick C1 protein: NPC1 involvement in late endocytic events. Mol Biol Cell. 12:601-14.
Ko, D.C., L. Milenkovic, S.M. Beier, H. Manuel, J. Buchanan, and M.P. Scott. 2005. Cell-autonomous death of cerebellar purkinje neurons with autophagy in Niemann-Pick type C disease. PLoS Genet. 1:81-95.
Kobayashi, T., M.H. Beuchat, M. Lindsay, S. Frias, R.D. Palmiter, H. Sakuraba, R.G. Parton, and J. Gruenberg. 1999. Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport. Nat Cell Biol. 1:113-8.
Kuwabara, P.E., and M. Labouesse. 2002. The sterol-sensing domain: multiple families, a unique role? Trends Genet. 18:193-201.
Lange, Y. 1991. Disposition of intracellular cholesterol in human fibroblasts. J Lipid Res. 32:329-39.
Larsen, L.B., P. Ravn, A. Boisen, L. Berglund, and T.E. Petersen. 1997. Primary structure of EPV20, a secretory glycoprotein containing a previously uncharacterized type of domain. Eur J Biochem. 243:437-41.
Lebrand, C., M. Corti, H. Goodson, P. Cosson, V. Cavalli, N. Mayran, J. Faure, and J. Gruenberg. 2002. Late endosome motility depends on lipids via the small GTPase Rab7. Embo J. 21:1289-300.
Liou, H.L., S.S. Dixit, S. Xu, G.S. Tint, A.M. Stock, and P. Lobel. 2006. NPC2, the protein deficient in Niemann-Pick C2 disease, consists of multiple glycoforms that bind a variety of sterols. J Biol Chem. 281:36710-23.
Liscum, L., and J.R. Faust. 1987. Low density lipoprotein (LDL)-mediated suppression of cholesterol synthesis and LDL uptake is defective in Niemann-Pick type C fibroblasts. J Biol Chem. 262:17002-8.
Liscum, L., and J.J. Klansek. 1998. Niemann-Pick disease type C. Curr Opin Lipidol. 9:131-5. Lloyd-Evans, E., A.J. Morgan, X. He, D.A. Smith, E. Elliot-Smith, D.J. Sillence, G.C. Churchill,
E.H. Schuchman, A. Galione, and F.M. Platt. 2008. Niemann-Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium. Nat Med. 14:1247-55.
Lombardi, D., T. Soldati, M.A. Riederer, Y. Goda, M. Zerial, and S.R. Pfeffer. 1993. Rab9 functions in transport between late endosomes and the trans Golgi network. Embo J. 12:677-82.
REFERENCES
55
Matter, K., J.A. Whitney, E.M. Yamamoto, and I. Mellman. 1993. Common signals control low density lipoprotein receptor sorting in endosomes and the Golgi complex of MDCK cells. Cell. 74:1053-64.
Millard, E.E., S.E. Gale, N. Dudley, J. Zhang, J.E. Schaffer, and D.S. Ory. 2005. The sterol-sensing domain of the Niemann-Pick C1 (NPC1) protein regulates trafficking of low density lipoprotein cholesterol. J Biol Chem. 280:28581-90.
Möbius, W., E. van Donselaar, Y. Ohno-Iwashita, Y. Shimada, H.F. Heijnen, J.W. Slot, and H.J. Geuze. 2003. Recycling compartments and the internal vesicles of multivesicular bodies harbor most of the cholesterol found in the endocytic pathway. Traffic. 4:222-31.
Muller, T., M.W. Hess, N. Schiefermeier, K. Pfaller, H.L. Ebner, P. Heinz-Erian, H. Ponstingl, J. Partsch, B. Rollinghoff, H. Kohler, T. Berger, H. Lenhartz, B. Schlenck, R.J. Houwen, C.J. Taylor, H. Zoller, S. Lechner, O. Goulet, G. Utermann, F.M. Ruemmele, L.A. Huber, and A.R. Janecke. 2008. MYO5B mutations cause microvillus inclusion disease and disrupt epithelial cell polarity. Nat Genet. 40:1163-5.
Munehira, Y., T. Ohnishi, S. Kawamoto, A. Furuya, K. Shitara, M. Imamura, T. Yokota, S. Takeda, T. Amachi, M. Matsuo, N. Kioka, and K. Ueda. 2004. Alpha1-syntrophin modulates turnover of ABCA1. J Biol Chem. 279:15091-5.
Mutka, A.L., S. Lusa, M.D. Linder, E. Jokitalo, O. Kopra, M. Jauhiainen, and E. Ikonen. 2004. Secretion of sterols and the NPC2 protein from primary astrocytes. J Biol Chem. 279:48654-62.
Nachury, M.V., A.V. Loktev, Q. Zhang, C.J. Westlake, J. Peranen, A. Merdes, D.C. Slusarski, R.H. Scheller, J.F. Bazan, V.C. Sheffield, and P.K. Jackson. 2007. A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell. 129:1201-13.
Narita, K., A. Choudhury, K. Dobrenis, D.K. Sharma, E.L. Holicky, D.L. Marks, S.U. Walkley, and R.E. Pagano. 2005. Protein transduction of Rab9 in Niemann-Pick C cells reduces cholesterol storage. Faseb J. 19:1558-60.
Naureckiene, S., D.E. Sleat, H. Lackland, A. Fensom, M.T. Vanier, R. Wattiaux, M. Jadot, and P. Lobel. 2000. Identification of HE1 as the second gene of Niemann-Pick C disease. Science. 290:2298-301.
Navab, M., J.A. Berliner, A.D. Watson, S.Y. Hama, M.C. Territo, A.J. Lusis, D.M. Shih, B.J. Van Lenten, J.S. Frank, L.L. Demer, P.A. Edwards, and A.M. Fogelman. 1996. The Yin and Yang of oxidation in the development of the fatty streak. A review based on the 1994 George Lyman Duff Memorial Lecture. Arterioscler Thromb Vasc Biol. 16:831-42.
Neufeld, E.B., A.T. Remaley, S.J. Demosky, J.A. Stonik, A.M. Cooney, M. Comly, N.K. Dwyer, M. Zhang, J. Blanchette-Mackie, S. Santamarina-Fojo, and H.B. Brewer, Jr. 2001. Cellular localization and trafficking of the human ABCA1 transporter. J Biol Chem. 276:27584-90.
Neufeld, E.B., J.A. Stonik, S.J. Demosky, Jr., C.L. Knapper, C.A. Combs, A. Cooney, M. Comly, N. Dwyer, J. Blanchette-Mackie, A.T. Remaley, S. Santamarina-Fojo, and H.B. Brewer, Jr. 2004. The ABCA1 transporter modulates late endocytic trafficking: insights from the correction of the genetic defect in Tangier disease. J Biol Chem. 279:15571-8.
Neufeld, E.B., M. Wastney, S. Patel, S. Suresh, A.M. Cooney, N.K. Dwyer, C.F. Roff, K. Ohno, J.A. Morris, E.D. Carstea, J.P. Incardona, J.F. Strauss, 3rd, M.T. Vanier, M.C. Patterson, R.O. Brady, P.G. Pentchev, and E.J. Blanchette-Mackie. 1999. The Niemann-Pick C1
MATTS LINDER
56
protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo. J Biol Chem. 274:9627-35.
Nofer, J.R., A.T. Remaley, R. Feuerborn, I. Wolinnska, T. Engel, A. von Eckardstein, and G. Assmann. 2006. Apolipoprotein A-I activates Cdc42 signaling through the ABCA1 transporter. J Lipid Res. 47:794-803.
Nohturfft, A., R.A. DeBose-Boyd, S. Scheek, J.L. Goldstein, and M.S. Brown. 1999. Sterols regulate cycling of SREBP cleavage-activating protein (SCAP) between endoplasmic reticulum and Golgi. Proc Natl Acad Sci U S A. 96:11235-40.
Ohgami, N., D.C. Ko, M. Thomas, M.P. Scott, C.C. Chang, and T.Y. Chang. 2004. Binding between the Niemann-Pick C1 protein and a photoactivatable cholesterol analog requires a functional sterol-sensing domain. Proc Natl Acad Sci U S A. 101:12473-8.
Ohsaki, Y., Y. Sugimoto, M. Suzuki, H. Hosokawa, T. Yoshimori, J.P. Davies, Y.A. Ioannou, M.T. Vanier, K. Ohno, and H. Ninomiya. 2006. Cholesterol depletion facilitates ubiquitylation of NPC1 and its association with SKD1/Vps4. J Cell Sci. 119:2643-53.
Okamura, N., S. Kiuchi, M. Tamba, T. Kashima, S. Hiramoto, T. Baba, F. Dacheux, J.L. Dacheux, Y. Sugita, and Y.Z. Jin. 1999. A porcine homolog of the major secretory protein of human epididymis, HE1, specifically binds cholesterol. Biochim Biophys Acta. 1438:377-87.
Okuhira, K., M.L. Fitzgerald, D.A. Sarracino, J.J. Manning, S.A. Bell, J.L. Goss, and M.W. Freeman. 2005. Purification of ATP-binding cassette transporter A1 and associated binding proteins reveals the importance of beta1-syntrophin in cholesterol efflux. J Biol Chem. 280:39653-64.
Omori, Y., C. Zhao, A. Saras, S. Mukhopadhyay, W. Kim, T. Furukawa, P. Sengupta, A. Veraksa, and J. Malicki. 2008. Elipsa is an early determinant of ciliogenesis that links the IFT particle to membrane-associated small GTPase Rab8. Nat Cell Biol. 10:437-44.
Osborne, T.F., and J.M. Rosenfeld. 1998. Related membrane domains in proteins of sterol sensing and cell signaling provide a glimpse of treasures still buried within the dynamic realm of intracellular metabolic regulation. Curr Opin Lipidol. 9:137-40.
Pacheco, C.D., R. Kunkel, and A.P. Lieberman. 2007. Autophagy in Niemann-Pick C disease is dependent upon Beclin-1 and responsive to lipid trafficking defects. Hum Mol Genet. 16:1495-503.
Patel, S.C., S. Suresh, U. Kumar, C.Y. Hu, A. Cooney, E.J. Blanchette-Mackie, E.B. Neufeld, R.C. Patel, R.O. Brady, Y.C. Patel, P.G. Pentchev, and W.Y. Ong. 1999. Localization of Niemann-Pick C1 protein in astrocytes: implications for neuronal degeneration in Niemann- Pick type C disease. Proc Natl Acad Sci U S A. 96:1657-62.
Pentchev, P.G., M.E. Comly, H.S. Kruth, M.T. Vanier, D.A. Wenger, S. Patel, and R.O. Brady. 1985. A defect in cholesterol esterification in Niemann-Pick disease (type C) patients. Proc Natl Acad Sci U S A. 82:8247-51.
Peränen, J., P. Auvinen, H. Virta, R. Wepf, and K. Simons. 1996. Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts. J Cell Biol. 135:153-67.
Pfeffer, S.R. 2001. Rab GTPases: specifying and deciphering organelle identity and function. Trends Cell Biol. 11:487-91.
Press, B., Y. Feng, B. Hoflack, and A. Wandinger-Ness. 1998. Mutant Rab7 causes the accumulation of cathepsin D and cation-independent mannose 6-phosphate receptor in an early endocytic compartment. J Cell Biol. 140:1075-89.
REFERENCES
57
Puri, V., R. Watanabe, M. Dominguez, X. Sun, C.L. Wheatley, D.L. Marks, and R.E. Pagano. 1999. Cholesterol modulates membrane traffic along the endocytic pathway in sphingolipid-storage diseases. Nat Cell Biol. 1:386-8.
Quinn, M.T., S. Parthasarathy, L.G. Fong, and D. Steinberg. 1987. Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocyte/macrophages during atherogenesis. Proc Natl Acad Sci U S A. 84:2995-8.
Radhakrishnan, A., Y. Ikeda, H.J. Kwon, M.S. Brown, and J.L. Goldstein. 2007. Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: oxysterols block transport by binding to Insig. Proc Natl Acad Sci U S A. 104:6511-8.
Radhakrishnan, A., L.P. Sun, H.J. Kwon, M.S. Brown, and J.L. Goldstein. 2004. Direct binding of cholesterol to the purified membrane region of SCAP: mechanism for a sterol-sensing domain. Mol Cell. 15:259-68.
Ren, M., G. Xu, J. Zeng, C. De Lemos-Chiarandini, M. Adesnik, and D.D. Sabatini. 1998. Hydrolysis of GTP on rab11 is required for the direct delivery of transferrin from the pericentriolar recycling compartment to the cell surface but not from sorting endosomes. Proc Natl Acad Sci U S A. 95:6187-92.
Riederer, M.A., T. Soldati, A.D. Shapiro, J. Lin, and S.R. Pfeffer. 1994. Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the trans-Golgi network. J Cell Biol. 125:573-82.
Roland, J.T., A.K. Kenworthy, J. Peranen, S. Caplan, and J.R. Goldenring. 2007. Myosin Vb interacts with Rab8a on a tubular network containing EHD1 and EHD3. Mol Biol Cell. 18:2828-37.
Rust, S., M. Rosier, H. Funke, J. Real, Z. Amoura, J.C. Piette, J.F. Deleuze, H.B. Brewer, N. Duverger, P. Denefle, and G. Assmann. 1999. Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1. Nat Genet. 22:352-5.
Sahlender, D.A., R.C. Roberts, S.D. Arden, G. Spudich, M.J. Taylor, J.P. Luzio, J. Kendrick-Jones, and F. Buss. 2005. Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis. J Cell Biol. 169:285-95.
Saito, H., P. Dhanasekaran, D. Nguyen, P. Holvoet, S. Lund-Katz, and M.C. Phillips. 2003. Domain structure and lipid interaction in human apolipoproteins A-I and E, a general model. J Biol Chem. 278:23227-32.
Saito, H., S. Lund-Katz, and M.C. Phillips. 2004. Contributions of domain structure and lipid interaction to the functionality of exchangeable human apolipoproteins. Prog Lipid Res. 43:350-80.
Sasaki, T., A. Kikuchi, S. Araki, Y. Hata, M. Isomura, S. Kuroda, and Y. Takai. 1990. Purification and characterization from bovine brain cytosol of a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to smg p25A, a ras p21-like GTP-binding protein. J Biol Chem. 265:2333-7.
Sato, T., S. Mushiake, Y. Kato, K. Sato, M. Sato, N. Takeda, K. Ozono, K. Miki, Y. Kubo, A. Tsuji, R. Harada, and A. Harada. 2007. The Rab8 GTPase regulates apical protein localization in intestinal cells. Nature. 448:366-9.
Schalk, I., K. Zeng, S.K. Wu, E.A. Stura, J. Matteson, M. Huang, A. Tandon, I.A. Wilson, and W.E. Balch. 1996. Structure and mutational analysis of Rab GDP-dissociation inhibitor. Nature. 381:42-8.
MATTS LINDER
58
Schrantz, N., Y. Sagiv, Y. Liu, P.B. Savage, A. Bendelac, and L. Teyton. 2007. The Niemann-Pick type C2 protein loads isoglobotrihexosylceramide onto CD1d molecules and contributes to the thymic selection of NKT cells. J Exp Med. 204:841-52.
Schwenke, D.C., and T.E. Carew. 1989. Initiation of atherosclerotic lesions in cholesterol-fed rabbits. I. Focal increases in arterial LDL concentration precede development of fatty streak lesions. Arteriosclerosis. 9:895-907.
Segev, N. 2001. Ypt and Rab GTPases: insight into functions through novel interactions. Curr Opin Cell Biol. 13:500-11.
Sever, N., T. Yang, M.S. Brown, J.L. Goldstein, and R.A. DeBose-Boyd. 2003. Accelerated degradation of HMG CoA reductase mediated by binding of insig-1 to its sterol-sensing domain. Mol Cell. 11:25-33.
Shapiro, A.D., and S.R. Pfeffer. 1995. Quantitative analysis of the interactions between prenyl Rab9, GDP dissociation inhibitor-alpha, and guanine nucleotides. J Biol Chem. 270:11085-90.
Sleat, D.E., J.A. Wiseman, M. El-Banna, S.M. Price, L. Verot, M.M. Shen, G.S. Tint, M.T. Vanier, S.U. Walkley, and P. Lobel. 2004. Genetic evidence for nonredundant functional cooperativity between NPC1 and NPC2 in lipid transport. Proc Natl Acad Sci U S A. 101:5886-91.
Song, B.L., N.B. Javitt, and R.A. DeBose-Boyd. 2005. Insig-mediated degradation of HMG CoA reductase stimulated by lanosterol, an intermediate in the synthesis of cholesterol. Cell Metab. 1:179-89.
Sugimoto, Y., H. Ninomiya, Y. Ohsaki, K. Higaki, J.P. Davies, Y.A. Ioannou, and K. Ohno. 2001. Accumulation of cholera toxin and GM1 ganglioside in the early endosome of Niemann-Pick C1-deficient cells. Proc Natl Acad Sci U S A. 98:12391-6.
Tang, C., A.M. Vaughan, G.M. Anantharamaiah, and J.F. Oram. 2006. Janus kinase 2 modulates the lipid-removing but not protein-stabilizing interactions of amphipathic helices with ABCA1. J Lipid Res. 47:107-14.
Tang, C., A.M. Vaughan, and J.F. Oram. 2004. Janus kinase 2 modulates the apolipoprotein interactions with ABCA1 required for removing cellular cholesterol. J Biol Chem. 279:7622-8.
Tanigawa, H., J.T. Billheimer, J. Tohyama, Y. Zhang, G. Rothblat, and D.J. Rader. 2007. Expression of cholesteryl ester transfer protein in mice promotes macrophage reverse cholesterol transport. Circulation. 116:1267-73.
Tontonoz, P., and D.J. Mangelsdorf. 2003. Liver X receptor signaling pathways in cardiovascular disease. Mol Endocrinol. 17:985-93.
Tsukamoto, K., K. Hirano, K. Tsujii, C. Ikegami, Z. Zhongyan, Y. Nishida, T. Ohama, F. Matsuura, S. Yamashita, and Y. Matsuzawa. 2001. ATP-binding cassette transporter-1 induces rearrangement of actin cytoskeletons possibly through Cdc42/N-WASP. Biochem Biophys Res Commun. 287:757-65.
Ullrich, O., S. Reinsch, S. Urbe, M. Zerial, and R.G. Parton. 1996. Rab11 regulates recycling through the pericentriolar recycling endosome. J Cell Biol. 135:913-24.
Ullrich, O., H. Stenmark, K. Alexandrov, L.A. Huber, K. Kaibuchi, T. Sasaki, Y. Takai, and M. Zerial. 1993. Rab GDP dissociation inhibitor as a general regulator for the membrane association of rab proteins. J Biol Chem. 268:18143-50.
Walkley, S.U., and K. Suzuki. 2004. Consequences of NPC1 and NPC2 loss of function in mammalian neurons. Biochim Biophys Acta. 1685:48-62.
REFERENCES
59
Walter, M., J.P. Davies, and Y.A. Ioannou. 2003. Telomerase immortalization upregulates Rab9 expression and restores LDL cholesterol egress from Niemann-Pick C1 late endosomes. J Lipid Res. 44:243-53.
van der Sluijs, P., M. Hull, P. Webster, P. Male, B. Goud, and I. Mellman. 1992. The small GTP-binding protein rab4 controls an early sorting event on the endocytic pathway. Cell. 70:729-40.
Wang, N., W. Chen, P. Linsel-Nitschke, L.O. Martinez, B. Agerholm-Larsen, D.L. Silver, and A.R. Tall. 2003. A PEST sequence in ABCA1 regulates degradation by calpain protease and stabilization of ABCA1 by apoA-I. J Clin Invest. 111:99-107.
Wang, N., D.L. Silver, P. Costet, and A.R. Tall. 2000. Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1. J Biol Chem. 275:33053-8.
Wang, X., H.L. Collins, M. Ranalletta, I.V. Fuki, J.T. Billheimer, G.H. Rothblat, A.R. Tall, and D.J. Rader. 2007. Macrophage ABCA1 and ABCG1, but not SR-BI, promote macrophage reverse cholesterol transport in vivo. J Clin Invest. 117:2216-24.
Watari, H., E.J. Blanchette-Mackie, N.K. Dwyer, J.M. Glick, S. Patel, E.B. Neufeld, R.O. Brady, P.G. Pentchev, and J.F. Strauss, 3rd. 1999a. Niemann-Pick C1 protein: obligatory roles for N-terminal domains and lysosomal targeting in cholesterol mobilization. Proc Natl Acad Sci U S A. 96:805-10.
Watari, H., E.J. Blanchette-Mackie, N.K. Dwyer, M. Watari, E.B. Neufeld, S. Patel, P.G. Pentchev, and J.F. Strauss, 3rd. 1999b. Mutations in the leucine zipper motif and sterol-sensing domain inactivate the Niemann-Pick C1 glycoprotein. J Biol Chem. 274:21861-6.
Vedhachalam, C., P.T. Duong, M. Nickel, D. Nguyen, P. Dhanasekaran, H. Saito, G.H. Rothblat, S. Lund-Katz, and M.C. Phillips. 2007. Mechanism of ATP-binding cassette transporter A1-mediated cellular lipid efflux to apolipoprotein A-I and formation of high density lipoprotein particles. J Biol Chem. 282:25123-30.
Vedhachalam, C., L. Liu, M. Nickel, P. Dhanasekaran, G.M. Anantharamaiah, S. Lund-Katz, G.H. Rothblat, and M.C. Phillips. 2004. Influence of ApoA-I structure on the ABCA1-mediated efflux of cellular lipids. J Biol Chem. 279:49931-9.
Wennerberg, K., K.L. Rossman, and C.J. Der. 2005. The Ras superfamily at a glance. J Cell Sci. 118:843-6.
Willenborg, M., C.K. Schmidt, P. Braun, J. Landgrebe, K. von Figura, P. Saftig, and E.L. Eskelinen. 2005. Mannose 6-phosphate receptors, Niemann-Pick C2 protein, and lysosomal cholesterol accumulation. J Lipid Res. 46:2559-69.
Williams, K.J., and I. Tabas. 1998. The response-to-retention hypothesis of atherogenesis reinforced. Curr Opin Lipidol. 9:471-4.
Wilson, A.L., R.A. Erdman, F. Castellano, and W.A. Maltese. 1998. Prenylation of Rab8 GTPase by type I and type II geranylgeranyl transferases. Biochem J. 333 (Pt 3):497-504.
Xu, S., B. Benoff, H.L. Liou, P. Lobel, and A.M. Stock. 2007. Structural basis of sterol binding by NPC2, a lysosomal protein deficient in Niemann-Pick type C2 disease. J Biol Chem. 282:23525-31.
Yang, T., P.J. Espenshade, M.E. Wright, D. Yabe, Y. Gong, R. Aebersold, J.L. Goldstein, and M.S. Brown. 2002. Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER. Cell. 110:489-500.
MATTS LINDER
60
Yoshimura, S., J. Egerer, E. Fuchs, A.K. Haas, and F.A. Barr. 2007. Functional dissection of Rab GTPases involved in primary cilium formation. J Cell Biol. 178:363-9.
Zerial, M., and H. McBride. 2001. Rab proteins as membrane organizers. Nat Rev Mol Cell Biol. 2:107-17.
Zhang, M., N.K. Dwyer, D.C. Love, A. Cooney, M. Comly, E. Neufeld, P.G. Pentchev, E.J. Blanchette-Mackie, and J.A. Hanover. 2001a. Cessation of rapid late endosomal tubulovesicular trafficking in Niemann-Pick type C1 disease. Proc Natl Acad Sci U S A. 98:4466-71.
Zhang, M., N.K. Dwyer, E.B. Neufeld, D.C. Love, A. Cooney, M. Comly, S. Patel, H. Watari, J.F. Strauss, 3rd, P.G. Pentchev, J.A. Hanover, and E.J. Blanchette-Mackie. 2001b. Sterol-modulated glycolipid sorting occurs in niemann-pick C1 late endosomes. J Biol Chem. 276:3417-25.