Cholesterol Biosynthesis and ER Stress in Peroxisome Deficiency

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BIOCHI4338_proof 9 November 2013 1/11
Biochimie xxx (2013) 1e11
Contents lists available at ScienceDirect
Biochimie
journal homepage: www.elsevier.com/locate/biochi
Review
Cholesterol biosynthesis and ER stress in peroxisome deciency

Q4
Phyllis L. Faust a, Werner J. Kovacs b, c, *

a
Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA
Institute of Molecular Health Sciences, ETH Zuerich, CH-8093 Zuerich, Switzerland
c
Competence Center for Systems Physiology and Metabolic Diseases, ETH Zuerich, CH-8093 Zuerich, Switzerland
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 June 2013
Accepted 22 October 2013
Available online xxx
Cholesterol biosynthesis is a multi-step process involving more than 20 enzymes in several subcellular
compartments. The pre-squalene segment of the cholesterol/isoprenoid biosynthetic pathway is localized in peroxisomes. This review intends to highlight recent ndings illustrating the important role
peroxisomes play in cholesterol biosynthesis and maintenance of cholesterol homeostasis. Disruption of
the Pex2 gene leads to peroxisome deciency and widespread metabolic dysfunction. The Pex2/ mouse
model for Zellweger syndrome enabled us to evaluate the role of peroxisomes in cholesterol biosynthesis. These studies have shown that Pex2/ mice exhibit low levels of cholesterol in plasma and liver.
Pex2/ mice were unable to maintain normal cholesterol homeostasis despite activation of SREBP-2, the
master transcriptional regulator of cholesterol biosynthesis, and increased protein levels and activities of
cholesterol biosynthetic enzymes. The SREBP-2 pathway remained activated even after normalization of
hepatic cholesterol levels in response to bile acid feeding as well as in extrahepatic tissues and the liver
of neonatal and longer surviving Pex2 mutants, where cholesterol levels were normal. Several studies
have shown that endoplasmic reticulum (ER) stress can dysregulate lipid metabolism via SREBP activation independently of intracellular cholesterol concentration. We demonstrated that peroxisome
deciency activates endoplasmic reticulum stress pathways in Pex2/ mice, especially the integrated
stress response mediated by PERK and ATF4 signaling, and thereby leads to dysregulation of the SREBP-2
pathway. Our ndings suggest that functional peroxisomes are necessary to prevent chronic ER stress
and dysregulation of the endogenous sterol response pathway. The constitutive activation of ER stress
pathways might contribute to organ pathology and metabolic dysfunction in peroxisomal disorder
patients.
2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Peroxisomes
Cholesterol
Isoprenoid
Pex2
SREBP-2
ER stress
Integrated stress response
Unfolded protein response
Zellweger syndrome
1. Introduction
Abbreviations: ACAT, acetoacetyl-CoA thiolase; ATF, activating transcription

factor; BA, bile acid; CHOP, C/EBP homologous protein; CoA, coenzyme A; eIF2a,
eukaryotic translation initiation factor 2; ER, endoplasmic reticulum; FDPS, farnesyl-diphosphate synthase; Grp, glucose-regulated protein; HDL, high-density lipoprotein; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; HMGCS, 3-hydroxy3-methylglutaryl-CoA synthase; IDI, isopentenyl-diphosphate isomerase; Insig, insulin-induced gene; IRE1, inositol-requiring enzyme-1; ISA, isotopomer spectral
analysis; ISR, integrated stress response; LDL, low-density lipoprotein; MID, mass
isotopomer distribution; MVK, mevalonate kinase; MVD, mevalonate-diphosphate
decarboxylase; Pex, peroxin; PERK, protein kinase RNA-like endoplasmic reticulum
kinase; PMVK, phosphomevalonate kinase; PPARa, peroxisome proliferatoractivated receptor alpha; SCAP, SREBP cleavage-activating protein; SREBP, sterol
regulatory element-binding protein; UPR, unfolded protein response; VLCFA, very
long-chain fatty acid; XBP1, X-box binding protein 1.
* Corresponding author. Institute of Molecular Health Sciences, ETH Zuerich,
Schafmattstrasse 22, HPL H16, CH-8093 Zuerich, Switzerland. Tel.: 41 44 633
3084; fax: 41 44 633 1357.
E-mail address: werner.kovacs@biol.ethz.ch (W.J. Kovacs).
Peroxisomes are ubiquitous and highly versatile organelles of

eukaryotic cells that have many metabolic functions, including boxidation and a-oxidation of fatty acids, ether-phospholipid synthesis,
cholesterol and isoprenoid metabolism, bile acid (BA) synthesis, and
metabolism of reactive oxygen species (ROS) [1e3]. Peroxisomes can
multiply by ssion of pre-existing ones [4] or develop de novo from the
endoplasmic reticulum (ER) [5]. Proteins involved in peroxisome
biogenesis, the peroxins, are encoded by PEX genes. Peroxins are
involved in 3 key stages of peroxisomal development: (i) formation of
the peroxisomal membrane, (ii) compartmentalization of peroxisomal matrix proteins, and (iii) peroxisome proliferation (growth and
division). Peroxisomal matrix proteins are synthesized on free cytosolic ribosomes and contain peroxisomal targeting signals (PTS),
either C-terminal tripeptide (PTS1) or N-terminal nonapeptide (PTS2),
which are conserved throughout evolution. PTS-containing proteins
are recognized by the cytoplasmic import receptors Pex5p and Pex7p,
0300-9084/$ e see front matter 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.biochi.2013.10.019
Please cite this article in press as: P.L. Faust, W.J. Kovacs, Cholesterol biosynthesis and ER stress in peroxisome deciency, Biochimie (2013),
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P.L. Faust, W.J. Kovacs / Biochimie xxx (2013) 1e11
FA -oxidation
Acetyl-CoA
Acetoacetyl-CoA
thiolase
Acetoacetyl-CoA
HMG-CoA
Acetoacetate
synthase
HMG-CoA lyase
+
HMG-CoA
Acetyl-CoA
Cytoplasm
HMG-CoA
HMG-CoA reductase
HMG-CoA lyase
ER
Peroxisomes
-oxidation of VLCFAs, LCFAs
?
Pathway 2
Acetyl-CoA
Acetoacetyl-CoA
thiolase
Acetoacetyl-CoA
HMG-CoA synthase
Mitochondria
Mevalonate
Acetyl-CoA
Acetoacetyl-CoA
thiolase
Acetoacetyl-CoA
HMG-CoA synthase
HMG-CoA
HMG-CoA reductase
Pathway 1
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Mevalonate
Mevalonate kinase
Mevalonate-5-P
Phosphomevalonate
kinase
Mevalonate-5-PP
Diphosphomevalonate
decarboxylase
IPP isomerase
Isopentenyl-PP
Dimethylallyl-PP
Isopentenyl-tRNA
Farnesyldiphosphate
synthase
Farnesyl-PP
Squalene synthase
ER
Farnesyl moieties
Geranylgeranyl moieties
Dolichol
Heme A
Ubiquinone
Squalene
Squalene epoxidase
Squalene epoxide
Oxidosqualene cyclase
Lanosterol
7-Dehydrocholesterol
Desmosterol
7-DHC
Desmosterol
reductase
reductase
Cholesterol
Membrane/rafts
Steroid hormones
Lipoproteins
Protein modification
Bile acid synthesis in the liver
Peroxisomes
C27 bile acids
C24 bile acids

BAAT
Conjugated
bile acids
Excretion in bile
Fig. 1. Subcellular localization of cholesterol biosynthesis in mammalian cells and proposed pathways of acetyl-CoA utilization. Peroxisomal enzymes can convert acetyl-CoA, which
is presumably derived from peroxisomal very long-chain fatty acid oxidation, to farnesyl diphosphate (FPP) via HMG-CoA and mevalonate (Pathway 1). Reactions between
mevalonate and FPP are assumed to be almost exclusively peroxisomal and with the exception of HMG-CoA reductase (HMGCR) functional peroxisomal targeting signals have been
identied in all of those enzymes. FPP leaves the peroxisomes and is converted either to cholesterol in the endoplasmic reticulum (ER) or to nonsterol isoprenoids. In parallel,
cytosolic acetyl-CoA is used by cytosolic acetoacetyl-CoA thiolase and HMG-CoA synthase to form HMG-CoA, which is converted to mevalonate by ER-localized HMGCR. ER-localized
HMGCR is bound to ER membranes through its NH2-terminal domain that contains eight transmembrane helices, and the COOH-terminal domain projects into the cytosol and
contains the entire catalytic activity [27]. The mevalonate diffuses into the peroxisomes and mixes with locally made mevalonate. A single gene encodes the mitochondrial and
peroxisomal acetoacetyl-CoA thiolase. Similarly both the cytosolic and peroxisomal HMG-CoA synthase are encoded by a single gene. In the liver, part of the acetyl groups derived
from peroxisomal oxidation are released as free acetate; however, the mechanism of the transfer of peroxisomal acetyl-CoA to the cytosol needs to be further studied. Isotopomer
spectral analysis of fatty acids and sterols suggests that acetyl-CoA, formed by peroxisomal b-oxidation of very long-chain fatty acids and medium-chain dicarboxylic acids is directly
channeled to cholesterol synthesis inside the peroxisomes (Pathway 1) without mixing with cytosolic acetyl-CoA transferred from mitochondria before being used for fatty acid and
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Table 1
Sequences of functional peroxisomal targeting signal (PTS) motifs in cholesterol biosynthetic enzymes.
Q3
Protein name
Gene symbol
Sequence of carboxy terminus

including PTS1 motif
Organisma
UniProt accession
Acetoacetyl-CoA thiolase
ACAT1
Acat1
Acat1
PMVK
Pmvk
Pmvk
IDI1
Idi1
Idi1
GGGASAMLIQKL
GGGASALLIEKL
GGGASAVLIEKL
LENLIEFIRSRL
LEHLLGFIQAKL
NLLEFIHAKLQR
NQFVDHEKIYRM
SPFVDHEKIHRL
SPFVDHEKIHRM
Hs
Mm
Rn
Hs
Mm
Rn
Hs
Mm
Rn
P24752
Q8QZT1
P17764
Q15126
Q9D1G2
Q5RK24
Q13907
P58044
O35760
Phosphomevalonate kinase
Isopentenyl-diphosphate isomerase
Protein name
Gene symbol
Amino-terminal
Organisma
UniProt accession
Hs
Mm
Rn
Hs
Mm
Rn
Hs
Mm
Rn
Hs
Mm
Rn
Hs
Mm
Rn
Q01581
Q8JZK9
P17425
Q03426
Q9R008
P17256
P53602
Q99JF5
Q62967
Q13907
P58044
O35760
P14324
Q920E5
P05369
PTS2 sequences
HMG-CoA synthase
Mevalonate kinase
Mevalonate-diphosphate
decarboxylase
Isopentenyl-diphosphate isomerase
Farnesyl-diphosphate synthase
HMGCS1
Hmgcs1
Hmgcs1
MVK
Mvk
Mvk
MVD
Mvd
Mvd
IDI1
Idi1
Idi1
FDPS
Fdps
Fdps
Consensus PTS1
Consensus PTS2
SVKTNLMQL
SVKSNLMQL
SVKSNLMQL
KVILHGEHA
KVILHGEHA
KVILHGEHA
SVTLHQDQL
SVTLHQDQL
SVTLHQDQL
HLDKQQVQL
HLDEKQVQL
NLDEKQVQL
NSDVYAQE
KLDAYNQE
KLDVHNQE
(S/C/A)(K/R/H)(L/M)
(R/K)(L/V/I)X5(H/Q)(L/A)
Bolded letters in the sequences of peroxisomal targeting signal motifs indicate consensus amino acids.
a
Hs, Homo sapiens; Rn, Rattus norvegicus; Mm, Mus musculus.
respectively, and targeted to the docking and translocation machinery

on the peroxisomal membrane [5]. The importance of peroxisomes for
cellular metabolism is illustrated by the marked abnormalities in brain
and systemic organs in peroxisome biogenesis disorders of the Zellweger spectrum in which functional peroxisomes are absent [6] or
disorders caused by single peroxisomal enzyme deciencies [7].
The isoprenoid biosynthetic pathway is unrivaled in nature for the
chemical diversity of compounds it produces (Fig. 1) [8]. Products
from this pathway include sterol isoprenoids, such as cholesterol, and
non-sterol isoprenoids, such as heme A, ubiquinone, dolichol, and
isopentenyl tRNA. Cholesterol is an essential lipid in vertebrate cell
membranes and an obligatory precursor of steroid hormones, bile
acids, and oxysterols [9,10]. The mevalonate pathway (or presqualene segment) of isoprenoid biosynthesis allows eukaryotic
cells to convert acetyl-CoA into farnesyl diphosphate (FPP) (Fig.1). FPP
is either used for cholesterol biosynthesis by squalene synthase,
which catalyzes the rst committed step of cholesterol biosynthesis,
or it is used as precursor for the synthesis of non-sterol isoprenoids
(Fig. 1). FPP may also be used either directly or after conversion into
geranylgeranyl diphosphate for protein prenylation [8].
2. Localization of the pre-squalene segment of the isoprenoid
biosynthetic pathway in peroxisomes
Numerous studies have shown that the early steps in the
isoprenoid biosynthetic pathway occur in peroxisomes [reviewed
in Ref. [2]] (Fig. 1). Furthermore, there is conclusive evidence that
the enzymes catalyzing the conversion of mevalonate to FPP are

exclusively peroxisomal. With the exception of 3-hydroxy-3methylglutaryl-coenzyme A (HMGCR), the enzymes contain
functional PTSs (Table 1). A single gene encodes the mitochondrial and peroxisomal acetoacetyl-CoA thiolase (ACAT1), which
catalyzes the rst reaction of isoprenoid biosynthesis. Similarly,
both the cytosolic and peroxisomal HMG-CoA synthase
(HMGCS1) are encoded by a single gene. HMGCR catalyzes the
rate-limiting reaction of isoprenoid biosynthesis and is localized
both in the endoplasmic reticulum and peroxisomes [2,11,12]. A
comprehensive analysis of completely sequenced eukaryotic genomes provided evidence for a large number of independent
duplications of HMGCR in all eukaryotic kingdoms, but not for a
second HMGCR gene in mammals [12]. Therefore, the peroxisomal HMGCR activity in mammals is most likely due to alternative targeting of the ER enzyme to peroxisomes by an as yet
uncharacterized mechanism.
After some reports questioned the role of peroxisomes in
cholesterol biosynthesis in humans [13e15], we re-investigated the
subcellular localization of the pre-squalene isoprenoid biosynthetic
enzymes in human cells by using new stable isotopic techniques
and data computations with isotopomer spectral analysis, in
combination with immunouorescence and cell permeabilization
techniques [11]. These studies conrmed previous data showing
peroxisomal localization of the early steps in the isoprenoid
biosynthetic pathway [i.e., ACAT1, HMGCR, mevalonate kinase,
phosphomevalonate kinase, isopentenyl-diphosphate isomerase,
cholesterol synthesis (Pathway 2). Blue fonts in italic indicate SREBP-2-regulated cholesterol biosynthetic enzymes. The primary C24eBAs are formed from cholesterol by a sequence
of enzymatic modications involving several enzymes and multiple subcellular compartments [65,66]. Microsomal and cytosolic enzymes modify the steroid nucleus, and the
oxidation of the sterol side chain occurs in the mitochondrion, generating C27eBA intermediates. C24eBAs are formed from the C27eBA intermediates by peroxisomal b-oxidation of
the side chain, followed by conjugation of the C24eBAs to glycine or taurine, catalyzed by the peroxisomal enzyme bile acid-coenzyme A:amino acid N-acyltransferase (BAAT).
Conjugated C24eBAs are then transported out of the peroxisome to the hepatocyte cytosol and subsequently secreted into the bile.
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farnesyl-diphosphate synthase]. Importantly, we obtained further

proof for the localization of the pre-squalene segment of cholesterol synthesis in peroxisomes by mass isotopomer distribution
(MID) analysis of fatty acids and sterols in HepG2 and Chinese
hamster ovary (CHO) cells incubated with [1,2,3,4-13C4]docosanoate or [U-13C12]dodecanedioate [11]. Acetyl-CoA derived from
peroxisomal b-oxidation of very long-chain fatty acids and
medium-chain dicarboxylic acids was preferentially channeled to
cholesterol synthesis inside the peroxisomes. The MIDs of lathosterol and cholesterol were determined, and lathosterol was the
analyte of choice, because, unlike cholesterol, its pool is small and
turns over rapidly. In contrast, fatty acids (myristate, palmitate,
stearate, and oleate) were not labeled from [1,2,3,4-13C4]docosanoate or [U-13C12]dodecanedioate, indicating that acetyl-CoA
derived from peroxisomal b-oxidation is not released from peroxisomes and mixing with the cytosolic acetyl-CoA transferred from
mitochondria before being used for fatty acid and cholesterol
synthesis.
Further proof of the peroxisomal localization of cholesterol
biosynthetic enzymes comes from studies by Aboushadi et al. [16]
and Weinhofer et al. [17]. These studies have demonstrated that
HMGCR localized to peroxisomes is more resistant to inhibition by
statins, a class of cholesterol-lowering drugs, than ER HMGCR.
Weinhofer et al. (2006) showed that lovastatin suppresses cholesterol biosynthesis from [1-14C]acetate and [1-14C]C8:0, but not from
[1-14C]C24:0 as precursor, indicating that [1-14C]acetyl-CoA units
generated by peroxisomal fatty acid b-oxidation do not leave peroxisomes but rather are used for cholesterol synthesis.
In addition, new studies in plants provide evidence that the
rst and the nal four steps of the pre-squalene segment of the
isoprenoid biosynthetic pathway are localized to peroxisomes,
reinforcing the description of peroxisomes as major organelles in
isoprenoid biosynthesis. Arabidopsis thaliana acetoacetyl-CoA
thiolase (ACAT) was recently identied as a peroxisomal protein by a proteomic approach [18], conforming to the data from
mammalian systems [19]. In addition, bioinformatic localization
predictors identied a PTS1-like C-terminal motif in ACAT [20].
Arabidopsis and other plant species contain two isopentenyldiphosphate isomerase (IDI) genes (IDI1 and IDI2) [21] which
are transcribed each as long and short isoforms. Sapir-Mir et al.
[20] showed that the long versions encoded by the full IDI gene
open reading frames (ORFs) are targeted to chloroplasts and
mitochondria, whereas the short versions that start from the
second Met codon of the gene ORF contain a C-terminal PTS1 (e
HKL) and are localized to peroxisomes. Furthermore, the IDI1
gene from Catharanthus roseus also produces long and short
transcripts. The short C. roseus IDI1 isoform contains a C-terminal
PTS1 (eHKL) and is targeted to peroxisomes, despite the presence of an additional isoleucine residue downstream of the PTS1
[22]. Bioinformatic localization predictors identied PTS2-like
motifs in A. thaliana HMGCS, mevalonate kinase (MVK), and
mevalonate-diphosphate decarboxylase (MVD) which are very
similar in sequence and position to their mammalian counterparts [20]. Simkin et al. [23] showed that phosphomevalonate
kinase (PMVK) and MVD from A. thaliana and C. roseus are
localized to peroxisomes. In contrast to mammalian PMVKs,
which contain a PTS1 (Table 1), PMVK from A. thaliana and
C. roseus contain a putative PTS2. Farnesyl-diphosphate synthase
(FDPS) from C. roseus and Dictyostelium discoideum is targeted to
peroxisomes [24,25]. D. discoideum FDPS contains a N-terminal
sequence of nine amino acids that closely resembles the
consensus PTS2. Besides mammals and plants, the early steps of
the isoprenoid biosynthetic pathway in trypanosomatid parasites
are localized within peroxisome-related microbodies called glycosomes [26].
In summary, these results clearly show that in mammals

the rst part of isoprenoid/cholesterol synthesis from acetyl-CoA
to FPP occurs in peroxisomes. Furthermore, the targeting of
enzymes of the pre-squalene segment to peroxisomes in plants,
D. discoideum, and trypanosomatid parasites conforms to the
mammalian data and highlights the central role of peroxisomes in
isoprenoid biosynthesis.
3. Regulation of cholesterol biosynthesis and sterol
regulatory element-binding proteins
Cellular cholesterol levels are tightly regulated and reect a
delicate balance between dietary uptake, endogenous de novo
synthesis, efux, and conversion of cholesterol to bile acids
[9,10,27]. Cells contain an elaborate feedback system that senses
cholesterol levels and they adjust their sterol content by both
transcriptional and posttranscriptional feedback systems that
regulate cholesterol metabolism. Multiple mechanisms for feedback control of cholesterol biosynthesis converge on the ratelimiting enzyme in the pathway, HMGCR. HMGCR regulation
takes place at the levels of transcription, translation, posttranslational modication, and degradation. Fine-tuning of
cholesterol biosynthesis via post-translational regulation of
HMGCR is achieved through Insig-1-dependent proteasomal
degradation, which is triggered by the accumulation of sterols in ER
membranes [28]. Mammalian cells also modulate HMGCR activity
by AMP-activated protein kinase (AMPK)-mediated phosphorylation [27,29]. AMPK phosphorylates a conserved serine (Ser872) in
the HMGCR active site and thereby decreases HMGCR activity.
HMGCR is also regulated at the level of translation by a non-sterol
isoprenoid, but the mechanism is not clear [29].
Central to the transcriptional control of the cholesterol biosynthetic pathway is the sterol regulatory element-binding protein
(SREBP) family of membrane-bound, basic helix-loop-helix leucine
zipper (bHLH-LZ) transcription factors (Fig. 2) [27]. Mammalian
cells produce three SREBP isoforms, called SREBP-1a, SREBP-1c, and
SREBP-2 [27]. SREBP-1a and -1c are produced from the same gene
by use of different promoters and alternative splicing. SREBP-1c
activates predominantly genes involved in the synthesis of fatty
acids and their incorporation into triglycerides and phospholipids,
whereas SREBP-2 activates primarily genes required for the uptake
and biosynthesis of cholesterol [27]. SREBP-1a is a potent activator
of all SREBP-responsive genes. SREBP-1a and SREBP-2 are the predominant isoforms of SREBP in most cultured cell lines, whereas
SREBP-1c and SREBP-2 predominate in the liver and other tissues
in vivo [27]. SREBPs reside as transcriptionally inactive precursor
proteins in the ER membrane by their interaction with SREBP
cleavage-activating protein (SCAP), which functions as a sterol
sensor, and Insigs (insulin-induced genes) (Fig. 2). In steroldepleted cells, SCAP escorts SREBPs from the ER to the Golgi
apparatus, where two proteases (Site-1 and Site-2 protease) act
sequentially to release the N-terminal transcription factor domain
that enters the nucleus and activates SRE (sterol regulatory
element)-containing genes (Fig. 2). Sterol-stimulated binding of
two ER membrane proteins, Insig-1 and Insig-2, to the sterolsensing domain of SCAP leads to ER retention of the SCAP/SREBP
complex [27]. The major differences between Insig-1 and Insig-2
relate to the regulation of their expression. Insig-1 is an obligatory SREBP target gene, whereas Insig-2 is expressed at a low but
constitutive level and is not regulated by SREBPs [27]. Insig-1
expression is low at high sterol levels and at this stage SREBP is
regulated by Insig-2 alone. However, when sterols are limiting,
Insig-1 is induced and regulates SREBP with a greater sensitivity to
low sterol levels. In this way Insigs control SREBPs over a wide
range of sterol concentrations (Fig. 3).
Q1
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WD C
bHLH
Low Sterol
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SSD
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bH
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SREBP-2
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bHLH
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SRE
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LDL receptor
SREBP-2
Insig-1
Cholesterol biosynthetic genes
S2P
bHLH
S1P
Mature SREBP-2
Nucleus
GOLGI
Fig. 2. Model for the sterol-mediated post-translational proteolysis and activation of SREBP-2. Regulation of SREBP-2 occurs at the level of SREBP-2 synthesis, proteolytic activation,
transcriptional activity, and degradation. Precursor SREBP-2 is synthesized and embedded in ER membranes as inactive transcription factor. Multiple signals regulate the ER-toGolgi transport and the proteolytic activation of SREBP-2 by controlling the expression and stability of Insig. In the presence of cholesterol or oxysterol, the ER-to-Golgi transport is inhibited and SCAP-SREBP-2 is retained in the ER. Binding of cholesterol to SCAP changes its conformation and triggers the binding of SCAP to Insig, an ER anchor protein.
When cellular sterol levels are low, the afnity between SCAP and Insig is reduced and these proteins dissociate from each other, whereupon Insig is ubiquitinated and degraded in
proteasomes. SCAP-SREBP-2 is loaded into COPII-coated vesicles through an interaction with Sec23/Sec24 and transported to the Golgi. Once in the Golgi, the N-terminal transcription factor domain of SREBP-2 is released from the membrane by two sequential proteolytic cleavage events mediated by the Site-1 (S1P) and Site-2 (S2P) proteases. The
nuclear form of SREBP-2 activates target genes such as the LDL receptor, the cholesterol biosynthetic enzymes, Insig-1, and SREBP-2 through binding to sterol regulatory element
(SRE) sequences in gene promoters. Abbreviations are as follows: SREBP-2, sterol regulatory element-binding protein-2; SRE, sterol regulatory element; SCAP, SREBP cleavageactivating protein; Insig, insulin-induced gene; S1P, Site-1 protease; S2P, Site-2 protease; COPII, coat protein complex II; bHLH, basic helix-loop-helix; LDL, low-density lipoprotein; WD, tryptophan-aspartate repeat.
4. Cholesterol biosynthesis in peroxisome biogenesis

disorders
Reduced plasma cholesterol levels have been consistently
found in Zellweger spectrum patients [2]. Studies examining
rates of cholesterol synthesis, protein levels and activities of
enzymes of the pre-squalene segment in broblasts from Zellweger spectrum patients and peroxisome-decient CHO cells
produced conicting results [2]. Decreased activities of HMGCR,
MVK, PMVK, MVD, IDI1, and FDPS had been measured in liver
biopsies of Zellweger spectrum patients. However, Hogenboom
et al. [30] showed that decreased enzyme activities in livers from
human Zellweger patients might reect the bad condition of the
livers, since in human patients the time between death and autopsy may vary between a few hours and 1 day and several
cholesterol biosynthetic enzymes are labile. The cholesterol
biosynthetic pathway has also been analyzed in peroxisomedecient Pex5/ mice. The cholesterol biosynthesis rate was
normal in immortalized as well as primary Pex5/ mouse broblasts [31]. Newborn Pex5/ mice had normal levels of
cholesterol in plasma, liver and brain [31] and the activities of
cholesterol biosynthetic enzymes were either normal or slightly
increased [30]. A detailed summary of studies investigating the
effect of peroxisomal dysfunction on cholesterol levels and
cholesterol biosynthesis in cells and tissues from patients of the
Zellweger spectrum disorders can be found in our review from
2002 [2].
5. Peroxisome-decient Pex2 knockout mice are a model to

study cholesterol homeostasis in peroxisome biogenesis
disorders
5.1. The genetic background affects survival of Pex2 knockout mice
The PEX2 protein (Pex2p) is a peroxisomal integral membrane
protein involved in the import of peroxisomal matrix proteins; its
absence in both patients with peroxisomal defects and Pex2/ mice
results in a lack of functional peroxisomes and abnormal peroxisomal biochemical parameters (i.e., increased levels of very longchain fatty acids and C27eBA intermediates, decreased levels of
mature C24eBAs and plasmalogens, and localization of catalase to
the cytosol) [32,33]. The Pex2 null allele has been bred on several
different mouse genetic backgrounds, which markedly affects the
survival of Pex2/ mice. Homozygous Pex2/ mice on a hybrid
C57BL/6129SvJ genetic background usually die on the day of birth
(P0) [32], whereas Pex2/ mice on a Swiss Webster129S6/SvEv
genetic background (SW/129) survive up to 6 weeks [33e35]. When
the Pex2 null allele is congenic on either a 129S6/SvEv (129), C57BL/6
or Swiss Webster genetic background, there is signicant loss of
homozygous mutants during embryogenesis, with only 20e50%
surviving to birth and all mutants invariably dying on the day of birth
[34]. Since the lifespan of patients of the Zellweger spectrum is also
variable and ranges from a few weeks to early adulthood, Pex2/
mice on different genetic backgrounds represent a good model to
study the pathogenesis of peroxisome biogenesis disorders.
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translation
eIF2
eIF2-P
GADD34
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translation
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target genes
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ERAD
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XBP1
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GADD34 feedback
Fig. 3. Model illustrating the relationship between peroxisome deciency, ER stress, and activation of the integrated stress response. Peroxisome deciency activates hepatic ER
stress pathways in Pex2/ mice, especially the integrated stress response mediated by PERK and ATF4 signaling. Several metabolic derangements in peroxisome-decient Pex2/
livers are likely to trigger ER stress, including perturbed ux of mevalonate metabolites, altered bile acid homeostasis, changes in fatty acid levels and composition, and oxidative
stress. ER stress leads to the activation of the unfolded protein response (UPR). The UPR is initiated by three ER transmembrane proteins: PERK, IRE1, and ATF6. The ER chaperone
GRP78 is normally bound to these ER stress sensors and keeps them inactive, but dissociates from them under ER stress conditions to deal with the accumulation of unfolded
proteins in the ER lumen. This dissociation leads to the activation of the three UPR pathways. Dissociation of GRP78 from PERK leads to its homodimerization that induces PERK
autophosphorylation and kinase domain activation. Activated PERK phosphorylates serine 51 on the a-subunit of eIF2 which by inhibiting general mRNA translation reduces the
workload of the ER. In contrast to inhibition of general mRNA translation, the PERK/eIF2a pathway stimulates the translation of several specic mRNAs containing multiple 50 upstream open reading frames, such as ATF4. Increased levels of the ATF4 transcription factor triggers the activation of a gene expression program referred to as the integrated stress
response. ATF4 activates genes that encode functions in ER protein folding, ERAD, nutrient uptake, amino acid biosynthesis and transportation, the antioxidant stress response,
autophagy, transcription factors, apoptosis, and GADD34. GADD34 acts as a negative regulator of the PERK pathway by dephosphorylating eIF2a. IRE1 is activated by homodimerization and autophosphorylation. IRE1-mediated unconventional splicing removes a 26-nucleotide intron from unspliced full-length Xbp1 (X-box-binding protein 1) mRNA to
generate XBP1s, encoding a transcription factor for expression of ER chaperones and genes involved in ERAD and lipid biosynthesis. ER stress leads to the release of ATF6 from GRP78
and its translocation to the Golgi complex, where it is cleaved sequentially by Site-1 and Site-2 proteases to produce the active transcription factor. This process is similar to the
activation of the SREBPs. ATF6 activates the transcription of genes encoding chaperones, ERAD, C/EBP homologous protein (CHOP), and XBP1.
5.2. Plasma lipid and tissue cholesterol levels in Pex2/ mice

We examined cholesterol homeostasis in both newborn (P0)
Pex2/ mice from the 129 strain as well as P0, postnatal day 9e10
(P9eP10), and P36 Pex2/ mice from the SW/129 strain [36e38]. In
addition, we investigated cholesterol homeostasis in BA-treated
SW/129 Pex2/ mice [37]. BA treatment prolonged postnatal survival of SW/129 Pex2/ mice [33,37]. Total plasma cholesterol and
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HDL cholesterol were reduced by 43% and 73%, respectively, in P9

Pex2/ mice [36,37]. BA feeding normalized total plasma cholesterol levels in Pex2/ mice and decreased plasma HDL cholesterol
levels both in control and Pex2/ mice. In comparison with early
postnatal SW/129 Pex2/ mice, the extent of reduction in total
plasma cholesterol levels was similar in newborn 129 and SW/129
Pex2/ mice [38]. In summary, plasma cholesterol levels are
signicantly reduced both in Pex2/ mice as well as in patients of
the Zellweger spectrum disorders.
The steady-state cholesterol content in P9 Pex2/ livers was
decreased by 40% relative to control mouse livers [36], and BA feeding
normalized cholesterol levels in livers of Pex2/ mice [37]. Surprisingly, the hepatic cholesterol level was similar in P36 control and
Pex2/ mice [37]. Interestingly, the hepatic levels of squalene, an
intermediate in the cholesterol biosynthetic pathway, were signicantly decreased both in P9 and P36 untreated and BA-fed Pex2/
mice despite normalization of the cholesterol content in BA-fed
Pex2/ mice [37]. Total cholesterol levels were similar in the brain,
kidney, spleen, heart, and lung of P10 untreated and BA-fed Pex2/
mice when compared with control mice [36,37]. Tissue cholesterol
levels were also similar in P0 control and Pex2/ mice [38].
5.3. Cholesterol biosynthesis in Pex2/ mice
The activities of HMGCR, IDI1, FDPS, and squalene synthase were
increased w10e18-fold in the liver of P9 Pex2/ mice [36]. BA
feeding signicantly attenuated, but did not completely normalize,
these enzyme activities in Pex2/ mice [37]. The activities of these
enzymes were similar in livers of P0 SW/129 Pex2/ mice
compared to controls; however, the enzyme activities were
signicantly increased in livers of newborn 129 Pex2/ mice
despite normal hepatic cholesterol levels [38]. The hepatic protein
levels of cholesterol biosynthetic enzymes were also signicantly
increased in Pex2/ mice [36e38], and the increase in protein
levels was signicantly greater than the increase in the activities of
these enzymes. We normalized the specic activities of cholesterol
biosynthetic enzymes to the enzyme protein content within each
liver homogenate to obtain an estimate of catalytic efciency. The
catalytic efciency of HMGCR, IDI1, and FDPS was signicantly
decreased in P0 and untreated as well as BA-fed early postnatal
Pex2/ mice, suggesting that loss of peroxisomal compartmentalization and/or ER abnormalities affect the functional efciency of
these enzymes [36e38].
An interesting and different regulatory pattern of cholesterol
enzyme activities could be observed in kidneys from untreated and
BA-fed Pex2/ mice: the activity of the rate-limiting enzyme
HMGCR was signicantly decreased, whereas activities of other
cholesterol biosynthetic enzymes were increased [36,37]. HMGCR
activity was also signicantly decreased in kidneys from P0 129
Pex2/ mice, whereas activities of IDI1, FDPS, and squalene synthase were unchanged [38].
We measured the rate of in vivo cholesterol synthesis in P9
untreated and BA-fed SW/129 Pex2/ mice using [3H]acetate. The
rate of cholesterol synthesis was markedly increased 13-fold in the
liver of P9 Pex2/ mice compared to controls [36], which occurred
even despite the reduced catalytic efciency of hepatic cholesterol biosynthetic enzymes. However, protein levels and activities
of these enzymes are still highly increased in Pex2/ mice vs.
controls, thus enabling a compensatory increased cholesterol synthetic rate that is nonetheless still unable to maintain hepatic
cholesterol homeostasis. Surprisingly, despite normal tissue
cholesterol levels the rate of cholesterol synthesis was also significantly increased in the spleen, lung, and heart of P9 Pex2/ mice
compared to controls (6-, 2.8- and 4-fold, respectively) [36].
Whereas control mice showed incomplete conversion of [3H]
acetate to cholesterol in the liver, with accumulation of radiolabeled squalene and other minor precursors, Pex2/ mice
exhibited nearly quantitative incorporation of [3H]acetate into
cholesterol with little or no appreciable accumulation of radiolabeled precursors. BA feeding reduced hepatic cholesterol synthesis in Pex2/ mice to control levels, whereas the rate of
cholesterol synthesis in the spleen, heart, and lungs of BA-fed
Pex2/ mice was attenuated compared with that in untreated
Pex2/ mice but still signicantly increased compared with BA-fed
control mice [37]. Interestingly, the rate of cholesterol synthesis
was signicantly decreased w2-fold in the brain and kidneys of P9
Pex2/ mice compared to controls [36,37]. The decreased rate of
cholesterol synthesis correlates with decreased HMGCR activity in
kidneys of Pex2/ mice.
5.4. Cholesterol biosynthetic genes in Pex2/ mice
The hepatic expression of genes encoding SREBP-2-regulated
cholesterol biosynthetic enzymes, Srebp-2, Insig-1, and Insig-2 was
signicantly increased in postnatal SW/129 Pex2/ mice compared
to controls [36]. BA feeding reduced the expression of these genes
signicantly in Pex2/ livers, but the mRNA levels were still
signicantly increased compared with BA-fed control mice [37]. In
addition, we observed a very similar mRNA up-regulation of Srebp2 and its target genes in livers of P0 Pex2/ mice from both 129 and
SW/129 strains despite normal cholesterol levels [38].
In summary, the mRNA, protein and activity levels of cholesterol biosynthetic enzymes as well as cholesterol synthesis from
acetate were signicantly increased in livers of Pex2/ mice. This
was orchestrated by an upregulation of the SREBP-2 pathway,
which was however not able to maintain normal cholesterol homeostasis as hepatic and plasma cholesterol levels were markedly
reduced in early postnatal Pex2/ mice. In addition, the SREBP-2
pathway was activated even when hepatic cholesterol levels were
normal, including in P0 and P36 Pex2/ mice as well as after
normalization of hepatic cholesterol levels in response to BA
feeding. In spleen, lung, and heart of Pex2/ mice, cholesterol
level was normal but the cholesterol synthesis rate was increased,
which was only partially normalized by BA feeding. The kidney
displays an unusual decrease in HMGCR activity in Pex2/ mice
that appears to be post-translational. These ndings demonstrate
signicant diversity in the sterol sensing mechanism and SREBP-2
pathway dysregulations among various organs in Pex2/ peroxisome-decient mice.
6. ER stress is a consequence of peroxisome deciency
The Insig-SCAP-SREBP-2 protein network that controls the level
of cholesterol resides in the ER. The ER membrane has very low
levels of sterols compared to the whole cell, making it a highly
effective environment for the high-afnity cholesterol sensor SCAP
to reside. Radhakrishnan et al. [39] demonstrated that ER cholesterol levels and SREBP-2 cleavage were discernable over a range of
cholesterol levels. Plotting nuclear SREBP-2 as a function of ER
cholesterol revealed that the response of the SCAP sensing system
was strikingly sigmoidal, showing a sharp, switch-like transition
from little to nearly maximal SREBP-2 cleavage at 5 mol% ER
cholesterol. The concentration of ER cholesterol that denes the
switching point is set by the ration of SCAP to Insig. Since SCAP is a
tetramer it has been hypothesized that binding of cholesterol to
one of the four binding sites in the SCAP tetramer increases the
afnity of the other sites for cholesterol, resulting in sharp cooperativity. Hence, cellular cholesterol levels are controlled with
remarkable precision by cooperative interactions between ER
cholesterol, SCAP, and Insig. The fact that the cholesterol transition/
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threshold is moveable (e.g., Insig overexpression) indicates that it

might also be altered by other means, such as small molecules,
dietary protocols, stress or disturbed ER homeostasis. Since
microsomal abnormalities have been observed previously in
peroxisome-decient hepatocytes in both Pex2/ [33] and Pex5/
mice [40], we hypothesized that ER dysfunction might disturb the
regulation of cholesterol biosynthesis in Pex2/ mice.
involving interferon, and HRI is activated by heme deprivation [42].

This pathway is referred to as the integrated stress response (ISR),
because divergent signals activate the four different eIF2a kinases
and the ISR maintains the balance between restoration of cellular
homeostasis under physiological stress and apoptosis.
6.1. Endoplasmic reticulum stress and the unfolded protein

response
Several studies have reported that ER stress and activation of the

UPR induce lipid dysregulation via SREBP-2 activation independently of intracellular cholesterol concentration [48e51]. SREBP-2
is activated during ER stress through the same pathway that is
triggered by sterol depletion: ER stress and induction of the UPR
results in Insig-1 dissociating from SCAP, whereupon Insig is
ubiquitinated and degraded in proteasomes, and SCAP-SREBP-2
moves to the Golgi. In addition, translation attenuation in ERstressed cells due to PERK activation also decreases protein levels
of Insig-1 [49]. Insig-2 cannot substitute for Insig-1 in blocking
SREBP cleavage under conditions of hypotonic stress [49].
Furthermore, Kammoun et al. [52] showed that GRP78 overexpression inhibits ER stress-induced SREBP-1c/2 activation in
hepatocytes and reduces hepatic steatosis in insulin-resistant mice.
Since Pex2/ mice displayed persistent up-regulation of cholesterogenic pathways in liver even when hepatic cholesterol
levels were normal, as seen in P0 and P36 mutants and in BA-fed
early postnatal mutants, we examined whether peroxisome deciency causes ER stress, activates the unfolded protein response
and thereby leads to SREBP-2 pathway activation. Accumulation of
the ER chaperone GRP78 was observed in variably sized cytosolic
aggregates in livers of untreated and BA-fed Pex2/ mice [37].
There was a consistent, strong induction of the integrated stress
response mediated by PERK and ATF4 signaling in livers of P0 and
postnatal Pex2/ mice from both strains [37,38]. The expression of
the transcriptional regulator p8 [stress-associated protein p8;
Nupr1 (nuclear protein-1)], a downstream target of the PERK
pathway, was highly up-regulated in livers of P0 129 and SW/129
Pex2/ mice and showed an ever increasing induction exacerbated by BA treatment in postnatal Pex2/ mice [37,38]. p8 is upregulated in response to several stresses, including lipopolysaccharide and proapoptotic agents [53], and our data suggest that
bile acids may also regulate this stress protein. At all ages, there
was absence of Xbp1 mRNA splicing, suggesting that IRE1 signaling
and its RNAse activity are not prominently induced in either P0 or
postnatal Pex2/ livers. The expression of genes encoding ER
chaperones and ERAD components in Pex2/ livers revealed an
evolving, age- and treatment-dependent pattern [37,38]. Evidence
for activation of the UPR was also found by microarray analysis in
adult Pex5-decient hepatocytes [54]. Our data suggest that the
induction of ER stress and SREBP-2 pathways occurs in parallel in
Pex2/ mice. For example, mRNA levels of SREBP-2 target genes
and ER stress markers were similar in P0 control and Pex2/ lung
and kidney, whereas their hepatic expression was signicantly
increased in Pex2/ mice. In addition, ER stress may be responsible for the decreased catalytic efciency of HMGCR in Pex2/
mice. It has been shown that tunicamycin, which inhibits protein
N-glycosylation and thereby induces ER stress, either inhibits
efcient induction of HMGCR activity in response to incubation in
lipoprotein-decient serum or decreases HMGCR activity under
steady state conditions [55].
The ER is the site of synthesis and folding of membrane and

secretory proteins. It is also a critical site of lipid and glucose
metabolism, calcium homeostasis, and detoxication of drugs and
metabolic byproducts. The term endoplasmic reticulum stress
denes any perturbation that compromises the protein folding
functionality of the ER. A number of biochemical and physiologic
stimuli, such as perturbation in calcium homeostasis or redox
status, elevated secretory protein synthesis, expression of misfolded proteins, sugar/glucose deprivation, altered glycosylation,
hypoxia, viral infection, and excess lipids can disrupt ER homeostasis and impose stress to the ER. These disturbances trigger an
evolutionary conserved response, termed the unfolded protein
response (UPR), in attempt to reestablish normal ER function.
The three canonical branches of the UPR are mediated by three
stress-sensing ER membrane-associated proteins: protein kinase
RNA-like ER kinase (PERK), inositol-requiring protein 1 (IRE1), and
activating transcription factor 6 (ATF6) [41e44]. Each ER stress
transducer denes a distinct arm of the UPR. In a stress-free ER,
these three transmembrane proteins are bound by the ER-resident
chaperone glucose-regulated protein of 78 kDa (GRP78) in their
intraluminal domains and rendered inactive [45,46]. Under ER
stress conditions GRP78 is required to deal with the accumulation
of unfolded proteins and dissociates from PERK, IRE1, and ATF6,
leading to their activation. IRE1 is a dual function serineethreonine
protein kinase and endoribonuclease. Activation of its RNase
domain results in the unconventional splicing of full-length XBP1
(X-box-binding protein 1) mRNA to generate spliced XBP1s. XBP1s
is a highly active transcription factor that activates genes that
enhance ER protein-folding capacity and degradation of misfolded
ER proteins. ATF6 is comprised of two isoforms, ATF6a and ATF6b,
and resides as transcriptionally inactive precursor proteins in the
ER membrane by its interaction with GRP78. ER stress and dissociation of GRP78 from ATF6 leads to its translocation to the Golgi
apparatus where it is cleaved sequentially by Site-1 and Site-2
proteases to produce an active transcription factor, a process
similar to that of the SREBPs. ATF6 induces XBP1 and genes mainly
encoding ER chaperones and proteins involved in ER-associated
degradation (ERAD).
Dissociation of GRP78 from PERK leads to its homodimerization
and activating autophosphorylation. PERK phosphorylates the
a-subunit of eukaryotic translation initiation factor 2 (eIF2a).
Phosphorylation of eIF2a at serine 51 reduces protein translation
and diminishes the workload of the ER [42,47]. Phosphorylated
eIF2a stimulates selective translation of the activating transcription
factor 4 (ATF4), which plays a crucial role for the adaptation to stress
[42]. The ATF4 target genes are involved in protein folding and assembly, metabolism, nutrient uptake, gene expression, alleviation of
oxidative stress, autophagy, and the regulation of apoptosis [42]. In
addition to PERK, three other kinases have been shown to induce
eIF2a phosphorylation and the preferential translation of ATF4:
GCN2 (general control non-derepressible kinase 2), PKR (doublestranded RNA-activated protein kinase), and HRI (heme-regulated
inhibitor kinase). GCN2 is the primary responder to nutritional
deprivation, PKR participates in an antiviral defense pathway
6.2. ER stress response and SREBP-2 activation
6.3. PPARa signaling, ER stress and the regulation of cholesterol

synthesis
Several studies have suggested an involvement of the
peroxisome proliferator-activated receptor alpha (PPARa) in the
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regulation of cholesterol synthesis; however, both stimulatory and

inhibitory effects of PPARa have been reported [56e62]. It has been
suggested that induction of PPARa leads to up-regulation of the
SREBP-2 pathway in livers of 2-day-old and adult mice with inactivation of the D-specic multifunctional protein 2 (Mfp2), which
catalyzes the second and third step in peroxisomal b-oxidation [62].
In addition, a recent study linked hepatocarcinogenesis in Acox1/
mice to ER stress mediated by sustained PPARa activation [63].
PPARa activation causes overexpression of the transcriptional
regulator p8, leading to an increase in ER stress effectors [63]. In
addition, it has been shown that all three isoforms of PPAR (a, g, and
d) interact with the p8 promoter to induce hepatic p8 gene
expression [63]. Potent endogenous PPARa ligands such as CoA
thioesters of very long-chain and branched-chain fatty acids are
metabolized in peroxisomes [64], and accumulation of these unmetabolized substrates in postnatal peroxisome-decient SW/129
Pex2/ livers hyperactivates PPARa, leading to increased expression of PPARa target genes (W.J. Kovacs and P.L. Faust, unpublished
results). However, the hepatic expression of PPARa and many PPARa
target genes was either unchanged or signicantly decreased in P0
129 and SW/129 Pex2/ mice [38], thus demonstrating that activation of SREBP-2 is not dependent on induction of PPARa pathways. Furthermore, treatment of early postnatal (2-week-old) wildtype mice with the peroxisome proliferator WY-14,643 strongly
induced hepatic PPARa target genes, but the expression of Srebp-2
and its target genes was reduced [38]. An inverse relationship between induction of PPARa-regulated genes and SREBP-2-regulated
genes is also seen in global gene expression proles in mouse liver
in fasting-to-feeding and feeding-to-fasting transitions (W.J.
Kovacs, unpublished results). In addition, as there is no activation of
either PPARa or PPARg (W.J. Kovacs, unpublished results) in P0
Pex2/ livers, clearly p8 is not an obligatory PPARa target for
activation of p8 or ER stress in peroxisome-decient mice.
6.4. Peroxisome deciency induces preferentially the integrated
stress response
How does peroxisome dysfunction lead to the ER stress
response? We hypothesize that several metabolic derangements in
peroxisome-decient Pex2/ liver are likely to trigger ER stress,
including perturbed ux of mevalonate metabolites, altered BA
homeostasis, changes in fatty acid levels and composition, and
oxidative stress. However, the multiple abnormalities at the
metabolite level in Pex2/ mice make it difcult to pinpoint in vivo
the exact mechanisms that trigger ER stress.
Dysregulation of BA homeostasis has been linked to ER stress
and UPR activation. It is well established that peroxisomes play a
crucial role in the synthesis of BAs. Cholesterol is converted into
BAs by a sequence of enzymatic modications involving several
enzymes and multiple subcellular compartments [65,66]. Microsomal and cytosolic enzymes modify the steroid nucleus, and the
oxidation of the sterol side chain occurs in the mitochondrion, the
end products of which are C27eBA intermediates. The nal conversion of C27eBA intermediates to the primary C24eBAs occurs in
the peroxisome, with shortening of the side chain by peroxisomal
b-oxidation, followed by conjugation of the C24eBAs to glycine or
taurine by the peroxisomal enzyme bile acid-coenzyme A:amino
acid N-acyltransferase (BAAT). The mature, conjugated C24eBAs
are then transported out of the peroxisome to the hepatocyte
cytosol and subsequently secreted into bile at the canalicular
membrane by BA transporters. Indeed, our studies suggest that BA
alterations could contribute to the activation of the ISR in Pex2/
mice. Cholestatic BA deposits were already observed in P0 Pex2/
livers, associated with very low BA concentration in bile and
canalicular damage, and BA measurements in liver and plasma
revealed an accumulation of mainly unconjugated C27eBAs and a

deciency of C24eBAs due to the biosynthesis defect [33]. In
addition, changes in expression of several BA transporters at the
basolateral membrane acted to limit the hepatic content of BAs
and their conjugates in Pex2/ mice, suggesting that the
peroxisome-decient liver is highly sensitive to bile acid toxicity
[33]. In a genetic model of intrahepatic cholestasis, the accumulation of BAs in the liver was associated with ER stress due to
cumulative defects in expression of bile acid-CoA ligase, involved
in conjugation of BAs returned to the liver via enterohepatic circulation, and BA transporters [67].
Alterations in cellular fatty acid composition induced by
peroxisome deciency may activate the ISR due to a disturbed
physical state of cellular membranes and altered function and/or
localization of membrane transport proteins. Peroxisome deciency leads to an accumulation of fatty acids that are degraded
via peroxisomal b-oxidation (e.g., very long-chain and branchedchain fatty acids, dicarboxylic acids). Furthermore, levels of some
n-6 polyunsaturated fatty acids were increased in the livers of P9
Pex2/ mice [37]. It has been shown that changes in fatty acid
composition in stearoyl-CoA desaturase-1-decient mice induce
ER stress [68] and that long-chain free fatty acids activate the UPR
in several cell types [69e71].
Peroxisomes play an important role in the homeostasis of
reactive oxygen species [3]. The ISR could be activated in Pex2/
mice through oxidative stress caused by defective peroxisomal
antioxidant mechanisms. Marked alterations of the mitochondria
were observed in both Pex2/ [33] mice and Pex5/ [72] mice,
which resemble closely those in disorders associated with oxidative
stress. The hepatic expression of the oxidative stress-activated
transcription factor Nrf2 (NF-E2-related factor 2) and several
target genes was highly increased in Pex2/ mice [37], suggesting
that increased oxidative stress is present in Pex2/ mice. However,
ER stress results also in the accumulation of ROS, and the PERK and
ATF4-mediated integrated stress response transcriptionally activates genes involved in amino acid metabolism and antioxidantdetoxifying enzymes that protect against oxidative stress [73].
Indeed, the hepatic expression of the amino acid biosynthetic
enzyme asparagine synthetase was highly increased in Pex2/
mice, whereas mRNA levels of amino acid catabolism genes were
highly decreased. Furthermore, PERK-mediated phosphorylation of
Nrf2 activates the antioxidant stress response and thereby connects
ER stress and oxidative stress signaling pathways.
Studies using genetic or dietary models of insulin resistance and
fatty liver have demonstrated a key interconnectedness between
hepatic steatosis and ER stress, as well as the physiological role of
the UPR sensors in lipid homeostasis [74]. UPR activation has been
observed in fatty liver diseases, although it is unclear how accumulation of excess lipids may engage ER stress response pathways
[75]. Alternatively, UPR activation could occur before the onset of
steatosis, and in fact steatosis may be a consequence of the UPR.
While hepatic steatosis could contribute to ER stress in postnatal
SW/129 Pex2/ mice, this is not the case in newborn Pex2/ mice.
ER stress pathways are induced in newborn Pex2/ liver in the
absence of hepatic steatosis [38].
Recent studies suggest that peroxins are inserted into the ER
during the biogenesis of peroxisomes [5,76], and retention of these
proteins in the ER due to disturbed peroxisome assembly in Pex2/
mice could induce ER stress and activate the UPR. However, our
studies suggest that peroxins are not retained in the ER and do not
contribute to induction of ER stress in Pex2/ liver. We found that
protein levels of several peroxins are reduced in the liver of both
newborn and postnatal Pex2/ mice [38]. The peroxins were only
found associated with peroxisomal membrane ghosts, which are
larger in size but fewer in number than normal peroxisomes.
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Perturbed levels of metabolites of the isoprenoid biosynthetic

pathway may activate the ISR in Pex2/ mice. For instance, it has
been shown that inhibition of cholesterol biosynthesis triggers the
UPR [77e79]. Perturbations of non-sterol isoprenoids downstream
of the farnesyl pyrophosphate branch point also contribute to the
induction of ER stress. Dolichol is involved in the N-linked glycosylation of ER proteins [8], and inhibition of N-linked glycosylation
by tunicamycin induces ER stress in a wide variety of cell types.
Perturbations in prenylated Rho and Rab proteins have been
demonstrated to result in ER stress [80,81]. Depletion of ubiquinone, a powerful antioxidant and important component of the
mitochondrial respiratory chain, may lead to oxidative stress and
subsequent ISR activation.
7. Conclusions and outlook
Several open questions remain to be addressed. Does ER stress
and UPR activation contribute to the in utero onset of pathology in
peroxisome biogenesis disorders? Preliminary results show that
the expression of SREBP-2 target genes and ER stress markers is
already increased in livers from embryonic day 18.5 SW/129
Pex2/ mice compared to controls (W.J. Kovacs and P.L. Faust,
unpublished results), but further studies are necessary to investigate the links between peroxisome deciency, cholesterol
biosynthesis, and UPR activation in fetal mice. Which pathogenic
factors (e.g. bile acid intermediate toxicity, very long-chain fatty
acid toxicity, plasmalogen deciency, oxidative stress, nonmetabolic factors) induce ER-associated stress pathways if peroxisomal metabolism is impaired? Why does peroxisome deciency
activate especially the integrated stress response mediated by
PERK and ATF4? Are ER stress pathways activated in the central
nervous system and contributing to neurological abnormalities
observed in peroxisome-decient mice and peroxisomal disorder
patients? Could patients with peroxisomal disorders benet from
therapies with chemical chaperones aiming to enhance the capacity of the ER and alleviate ER stress? What is the mechanism
that decreases HMGCR activity in peroxisome-decient Pex2/
kidneys? ER stress is associated with a range of diseases, including
ischemia/reperfusion injury, neurodegeneration, dyslipidemia, and
diabetes, making ER stress a probable instigator of pathological
cell death and dysfunction. Our ndings suggest that functional
peroxisomes are necessary to prevent chronic ER stress and dysregulation of the endogenous sterol response pathway. The
constitutive activation of ER stress pathways might aggravate the
phenotype in peroxisomal disorder patients. Therefore, we hypothesize that the study of ER-associated stress pathways in
peroxisomal disorders will lead to new perspectives and a better
understanding of the pathogenic mechanism of peroxisomal
disorders.
Acknowledgments
The authors wish to gratefully acknowledge the collaboration
and encouragement along the years of Dr. Skaidrite Krisans at San
Diego State University and Dr. Steven Fliesler at Research Service,
Veterans Administration Western New York Healthcare System and
University at Buffalo/State University of New York. We thank Dr.
Herbert Stangl for helpful discussions. The work was supported in
part by the Swiss National Science Foundation (SNF) grant
31003A_132982 to W.J.K.
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Cholesterol Biosynthesis and ER Stress in Peroxisome Deficiency

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Cholesterol Biosynthesis and ER Stress in Peroxisome Deficiency

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BIOCHI4338_proof 9 November 2013 1/11

Biochimie xxx (2013) 1e11

Contents lists available at ScienceDirect

Cholesterol biosynthesis and ER stress in peroxisome deciency

Phyllis L. Faust a, Werner J. Kovacs b, c, *

Abbreviations: ACAT, acetoacetyl-CoA thiolase; ATF, activating transcription

Peroxisomes are ubiquitous and highly versatile organelles of

P.L. Faust, W.J. Kovacs / Biochimie xxx (2013) 1e11

-oxidation of VLCFAs, LCFAs

BIOCHI4338_proof 9 November 2013 2/11

Bile acid synthesis in the liver

C24 bile acids

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P.L. Faust, W.J. Kovacs / Biochimie xxx (2013) 1e11

Sequence of carboxy terminus

respectively, and targeted to the docking and translocation machinery

the enzymes catalyzing the conversion of mevalonate to FPP are

BIOCHI4338_proof 9 November 2013 4/11

P.L. Faust, W.J. Kovacs / Biochimie xxx (2013) 1e11

farnesyl-diphosphate synthase]. Importantly, we obtained further

In summary, these results clearly show that in mammals

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P.L. Faust, W.J. Kovacs / Biochimie xxx (2013) 1e11

4. Cholesterol biosynthesis in peroxisome biogenesis

5. Peroxisome-decient Pex2 knockout mice are a model to

P.L. Faust, W.J. Kovacs / Biochimie xxx (2013) 1e11

BIOCHI4338_proof 9 November 2013 6/11

Integrated stress response

5.2. Plasma lipid and tissue cholesterol levels in Pex2/ mice

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HDL cholesterol were reduced by 43% and 73%, respectively, in P9

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P.L. Faust, W.J. Kovacs / Biochimie xxx (2013) 1e11

threshold is moveable (e.g., Insig overexpression) indicates that it

involving interferon, and HRI is activated by heme deprivation [42].

6.1. Endoplasmic reticulum stress and the unfolded protein

Several studies have reported that ER stress and activation of the

The ER is the site of synthesis and folding of membrane and

6.2. ER stress response and SREBP-2 activation

6.3. PPARa signaling, ER stress and the regulation of cholesterol

BIOCHI4338_proof 9 November 2013 9/11

P.L. Faust, W.J. Kovacs / Biochimie xxx (2013) 1e11

regulation of cholesterol synthesis; however, both stimulatory and

revealed an accumulation of mainly unconjugated C27eBAs and a

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Perturbed levels of metabolites of the isoprenoid biosynthetic

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deciency of enzymes involved in cholesterol biosynthesis, J. Lipid Res. 43

[55] J.J. Volpe, R.I. Goldberg, Effect of tunicamycin on 3-hydroxy-3-methylglutaryl

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