Download Mislocalization and inhibition of acetyl

Biochem. J. (2012) 448, 409–416 (Printed in Great Britain)
409
doi:10.1042/BJ20121158
Mislocalization and inhibition of acetyl-CoA carboxylase 1 by a synthetic
small molecule
Dongju JUNG*1 , Lutfi ABU-ELHEIGA†, Rie AYUZAWA*, Ziwei GU†, Takashi SHIRAKAWA‡, Yukio FUJIKI§, Norio NAKATSUJI*,
Salih J. WAKIL† and Motonari UESUGI*‡1
*Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto 606-8501, Japan, †Verna and Marrs McLean Department of Biochemistry and Molecular Biology,
Baylor College of Medicine, Houston, TX 77030, U.S.A., ‡Institute for Chemical Research, Kyoto University, Uji 611-0011, Japan, and §Department of Biology, Kyushu University
Faculty of Science, Fukuoka 812-81, Japan
Chromeceptin is a synthetic small molecule that inhibits insulininduced adipogenesis of 3T3-L1 cells and impairs the function of
IGF2 (insulin-like growth factor 2). The molecular target
of this benzochromene derivative is MFP-2 (multifunctional
protein 2). The interaction between chromeceptin and MFP-2
activates STAT6 (signal transducer and activator of transcription
6), which subsequently induces IGF inhibitory genes. It was not
previously known how the binding of chromeceptin with MFP-2
blocks adipogenesis and activates STAT6. The results of the
present study show that the chromeceptin–MFP-2 complex binds
to and inhibits ACC1 (acetyl-CoA carboxylase 1), an enzyme
important for the de novo synthesis of malonyl-CoA and fatty
acids. The formation of this ternary complex removes ACC1
from the cytosol and sequesters it in peroxisomes under the
guidance of Pex5p (peroxisomal-targeting signal type 1 receptor).
As a result, chromeceptin impairs fatty acid synthesis from
acetate where ACC1 is a rate-limiting enzyme. Overexpression of
malonyl-CoA decarboxylase or siRNA (small interfering RNA)
knockdown of ACC1 results in STAT6 activation, suggesting
a role for malonyl-CoA in STAT6 signalling. The molecular
mechanism of chromeceptin may provide a new pharmacological
approach to selective inhibition of ACC1 for biological studies
and pharmaceutical development.
INTRODUCTION
ACCs and MCD (malonyl-CoA decarboxylase), an enzyme
that converts malonyl-CoA into acetyl-CoA, regulate the cellular
concentrations of malonyl-CoA in response to nutritional and
hormonal conditions. Owing to their critical roles in the de
novo synthesis and oxidation of fatty acids, ACCs have been
considered as potential targets for pharmacological intervention of
obesity and cancer. For example, the synthetic small molecule CP610431 and its improved analogue CP-640186 inhibit the activity
of both ACC isoforms, block fatty acid synthesis and stimulate
fatty acid oxidation [11]. Another potent inhibitor of ACCs,
soraphen A, arrests the growth of prostate cancer cells [12].
Through phenotypic screening of a chemical library, we
previously discovered a synthetic small molecule that exhibits
cellular phenotypes similar to those of the ACC-targeting small
molecules [13]. Chromeceptin (Figure 1), a benzochromene
derivative, inhibits insulin-induced adipogenesis and selectively
reduces the growth and viability of human hepatocellular
carcinoma cells that overexpress IGF2 (insulin-like growth
factor-2) [13]. The results of mechanistic analyses suggest that
chromeceptin induces expression of IGF inhibitory genes, such
as IGFBP-1 (IGF-binding protein 1) and SOCS3 (suppressors
of cytokine signalling 3), through activation of STAT6 (signal
transducer and activator of transcription 6) [14].
Chromeceptin does not activate STAT6 through direct
interaction, but by binding to MFP-2 (multifunctional protein
2), also called D-bifunctional protein or 17-β-estradiol
dehydrogenase type IV. MFP-2 localizes to peroxisomes, due
ACCs (acetyl-CoA carboxylases, EC 6.4.1.2.) are biotincontaining enzymes involved in fatty acid biosynthesis and
oxidation [1,2]. ACCs catalyse the carboxylation of acetyl-CoA
to malonyl-CoA, a rate-limiting step in fatty acid biosynthesis,
through two enzymatic steps, biotin carboxylation and carboxyl
group transfer. Two isoforms of ACC have been identified in
mammals: ACC1 (ACCα, 265 kDa) and ACC2 (ACCβ, 280 kDa)
[3–6]. The two isoforms have the same enzymatic function and
similar amino-acid sequences, except for the 114 amino acids of
the N-terminus of ACC2, which contain a hydrophobic peptide
segment for mitochondrial membrane localization [7]. Despite
their similarities, ACC1 and ACC2 play different roles in fatty
acid metabolism, due to distinct subcellular localization and tissue
distribution. ACC1 is mainly expressed in the cytosol of cells
in lipogenic tissues, such as liver, adipose tissue and lactating
mammary gland. ACC2 is localized in the mitochondria, primarily
in tissues where fatty acid oxidation occurs, such as liver, muscle
and heart [6,7]. The different roles for ACC1 and ACC2 have been
confirmed in knockout mouse models, and are consistent with
their patterns of expression and localization. ACC1 contributes to
the de novo synthesis of fatty acids required for developmental
processes [8]. ACC2 negatively regulates fatty acid β-oxidation
in mitochondria with its enzymatic product, malonyl-CoA, which
is a potent inhibitor of CPT1 (carnitine palmitoyltransferase 1)
[9,10].
Key words: acetyl-CoA carboxylase (ACC), adipogenesis,
chromeceptin, malonyl-CoA, multifunctional protein 2 (MFP-2),
peroxisomal translocation.
Abbreviations used: ACC, acetyl-CoA carboxylase; BC, biotin carboxylase; CHO, Chinese-hamster ovary; CT, carboxyltransferase; DMEM, Dulbecco’s
modified Eagle’s medium; FKBP, FK506-binding protein; IGF, insulin-like growth factor; IL-4, interleukin-4; IP, immunoprecipitation; MCD, malonylCoA decarboxylase; MFP-2, multifunctional protein-2; PCC, propionyl-CoA carboxylase; Pex5p, peroxisomal-targeting signal type 1 receptor; pmp70,
peroxisomal membrane protein 70; PTS1, peroxisomal-targeting signal type 1; SEAP, secreted alkaline phosphatase; siRNA, small interfering RNA; STAT6,
signal transducer and activator of transcription 6.
1
Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
410
D. Jung and others
from Cell Signaling Technology. Anti-ACC antibodies
were from U.S. Biological and Cell Signaling Technology.
Anti-pmp70 (peroxisomal membrane protein 70) antibody and
other fluorescence-labelled antibodies were from Invitrogen
Life Technologies. The cDNA of MCD was from Origene.
[1-14 C]acetic acid was from MP Biochemicals. siRNA (small
interfering RNA) of ACC1 and a plasmid encoding a siRNA of
PCC (propionyl-CoA carboxylase) α were purchased from Santa
Cruz Biotechnology and Origene respectively.
Carboxylase assay
Figure 1
IL-4
Distinct mechanisms of STAT6 activation by chromeceptin and
The chemical structure of chromeceptin and the design of a STAT6-responsive reporter gene are
shown at the top. (A) Effects of chromeceptin on activation of the reporter gene in HepG2
cells. (B) Additive effect of IL-4 and 2 μM chromeceptin (Chrom) on STAT6 activation.
(C) Failure of 1 μM chromeceptin to induce phosphorylation of Tyr641 during 0.5–12 h incubations, compared with IL-4. Sample loading was normalized to actin level. (D) Failure of chromeceptin to activate the STAT6-responsive reporter gene in MFP-2 knockdown cells, compared
with 10 or 100 ng/ml IL-4 (means +
− S.E.M., n = 3 experiments). Inset: knockdown levels of
MFP-2 in the stably transfected cells. Con and KO indicate cell lines expressing an empty
plasmid and a plasmid expressing siRNA of MFP-2 respectively. Error bars indicate S.E.M. p-,
phospho-.
to the presence of the C-terminal PTS1 (peroxisomal-targeting
signal type 1), which is composed of an Ala-Lys-Leu amino-acid
sequence [15]. Without the PTS1 signal, MFP-2 localizes in the
cytosol, instead of in peroxisomes [16]. MFP-2 plays an important
role in β-oxidation of long-chain fatty acids [15]. MFP-2 knockout
mice accumulated immature bile acids in bile, and very-longchain fatty acids in brain and liver, whereas mitochondrial βoxidation was normal or even increased [17], suggesting that
MFP-2 has essential roles in the maturation of bile acids and
peroxisomal β-oxidation. The failure of chromeceptin to activate
STAT6 in a MFP-2 knockdown cell line confirmed the role of
MFP-2 in chromeceptin-induced STAT6 activation [14].
The results of the present study provide an insight into
how the chromeceptin–MFP-2 complex blocks insulin-induced
adipogenesis and activates STAT6. The complex recruits ACC1
from the cytosol to peroxisomes, impairs the ability of ACC1 to
synthesize malonyl-CoA in cytosol, and subsequently activates
STAT6 in a way that is different from activation by IL-4
(interleukin-4). The selective translocation of ACC1 by
chromeceptin may provide a new pharmacological approach to
reducing fat accumulation and to treating cancers in which there
is excessive fatty acid synthesis.
EXPERIMENTAL
Materials
Chromeceptin and anti-actin antibody were purchased from
Sigma–Aldrich. Anti-phospho-STAT6 (Tyr641 ) antibody was
c The Authors Journal compilation c 2012 Biochemical Society
HepG2 cells were cultured in DMEM (Dulbecco’s modified
Eagle’s medium) containing 10 % fetal bovine serum and
antibiotics. When the cells were confluent, the medium was
changed to a serum-free medium containing 1 μM chromeceptin
or 0.1 % DMSO. After a 6 h incubation, the cells were washed
twice with ice-cold PBS, collected and then extracted with
M-PER mammalian protein extraction reagent (Thermo
Scientific) in the presence of inhibitors for proteinases and
phosphatases (Roche). ACC activity in the extracts was monitored
as described previously [18]. Briefly, equal amounts of cell
extract were incubated with 0.4 mM acetyl-CoA or propionylCoA for 5 min to measure enzymatic activity of ACC or PCC
respectively. The amount of incorporated radiocarbon, from
sodium [14 C]bicarbonate, into enzymatic products of malonylCoA, by ACC, or methyl malonyl-CoA, by PCC, was measured
using a liquid scintillation counter, after removal of free
radiocarbon.
De novo fatty acid synthesis by acetate incorporation
HepG2 cells were cultured in DMEM containing 10 % fetal
bovine serum and antibiotics. When the cells were confluent,
the medium was changed to a serum-free medium containing
1 μM chromeceptin or 0.1 % DMSO. After 6 h of incubation,
1 μCi [14 C]acetic acid was added to the medium for an additional
12 h incubation. The cells were then washed with PBS 3–4 times
and the saponifiable (fatty acids) and unsaponifiable (cholesterol)
lipids were quantified as reported previously [19].
Transient co-transfection assay
The DNAs used in transfection assays were purified using
a Midi-Prep kit (Qiagen). HepG2 cells were plated out 24 h
before transfection with FuGENETM 6 (Roche). At 18 h after
transfection, the culture medium was changed to a serum-free
medium containing 0.5–5 μM chromeceptin or 10 ng/ml IL-4
(Promega). After a 20 h incubation, the activity of the secreted
alkaline phosphatase in the medium was measured using a Great
EscAPeTM Chemiluminescence kit (Clonetech), following the
manufacturer’s protocol. Each experiment was repeated three
times. For the siRNA experiments, 100 nM si-ACC1 or 1 μg of a
plasmid encoding si-PCCα was transfected, along with 0.5 μg of
the reporter gene, to HepG2 cells in a 24-well plate.
Immunoprecipitaion of MFP-2 and ACC1
HepG2 cells were treated for 6 h with 0.1 % DMSO or 1 μM
chromeceptin in a serum-free DMEM. After washing with icecold PBS, the cells were lysed in an IP (immunoprecipitation)
buffer [20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 % Nonidet
P40, 1 mM DTT (dithiothreitol) and 1 mM EDTA] containing a
mixture of inhibitors for proteinases and phophatases (Roche).
The soluble extracts were collected by ultracentrifugation,
pretreated with Protein G beads (Invitrogen), and stored in aliquots
at − 70 ◦ C. For IP, 10 mg of extract was incubated for 4 h at 4 ◦ C
Chromeceptin inhibits acetyl-CoA carboxylase 1
with 20 μg of an anti-MFP-2 antibody, raised in rabbit against the
C-terminal 15 amino acids (CSQKLQMILKDYAKL) of human
MFP-2 protein (NCBI Reference Sequence NP_000405.1).
The immune complex was precipitated with Protein A beads,
which were washed three times with the ice-cold IP buffer.
The bound proteins were eluted by boiling the beads in 1×
Laemmli sample buffer, separated on SDS/PAGE (4–12 %
gradient gel) (Invitrogen) and stained with Coomassie Blue G250 (Sigma). The prominent bands on the gel were excised for
peptide sequencing using a mass spectrophotometer, as described
previously [20]. Similarly, ACC1 was immunoprecipitated with
an anti-ACC1 antibody (Cell Signaling Technology) and Protein
A/G beads (Thermo Scientific). The bound proteins were eluted
and separated on SDS/PAGE as described for MFP-2 IP.
The separated proteins were transferred on to nitrocellulose
membranes and detected using Western bloting.
Immunohistochemistry
HepG2 cells and CHO (Chinese-hamster ovary) cells [wild type
(CHO-K1) and a mutant (CHO-ZP105)] were plated on to a 0.1 %
gelatin-coated glass plate with chambers (Electron Microscopy
Sciences) 24 h before treatment. The cells in each chamber were
treated for 1 h with 1 μM chromeceptin or 50 ng/ml IL-4 in a
serum-free medium. After fixing with 4 % paraformaldehyde
solution (Thermo Scientific), the cells were permeabilized with
1 % Triton X-100 solution. Rabbit anti-ACC1, anti-MFP2, antiACC2 and anti-pmp70 antibodies were labelled with a Zenon
Labeling kit (Invitrogen). Fluorescent images were captured and
analysed using a FluoViewTM confocal microscope (Olympus).
RESULTS
Activation of STAT6 by chromeceptin differs from activation by IL-4
We previously showed that chromeceptin activates the reporter
gene in which expression of the SEAP (secreted alkaline
phosphatase) gene is controlled by three copies of a STAT6binding site. In the present study, we used the same reporter
gene to compare the action of chromeceptin with that of IL-4, a
canonical activator of STAT6. The reporter gene was transiently
transfected into human hepatocellular carcinoma HepG2 cells,
and the expression of the reporter gene was evaluated in the
presence or absence of chromeceptin by measuring SEAP activity.
The activation of the reporter gene reached a maximum level at
2 μM chromeceptin. The maximum activity with chromeceptin
was 5.3-fold higher than activity without chromeceptin, and
was comparable with the activation level induced by 100 ng/ml
IL-4 (Figures 1A and 1B). When the cells were treated with both
2 μM chromeceptin and 10 or 100 ng/ml IL-4, activation of the
reporter gene was higher than the maximum level obtained with
chromeceptin alone, suggesting that chromeceptin and IL-4 have
different mechanisms of STAT6 activation. We also examined
the ability of chromeceptin to phosphorylate Tyr641 of STAT6, an
amino acid that is phosphorylated by IL-4. HepG2 cells treated
with chromeceptin for up to 12 h showed no phosphorylation of
Tyr641 , whereas IL-4 treatment induced phosphorylation of Tyr641
in 30 min (Figure 1C).
We previously demonstrated that MFP-2 plays an indispensible
role in chromeceptin-induced activation of STAT6 [14]. In the
present study, we examined the role of MFP-2 in IL-4-induced
STAT6 activation, using MFP-2 knockdown HepG2 cells in which
the expression of MFP-2 was reduced by a stably transfected
plasmid expressing siRNA of MFP-2. Although IL-4 was capable
of activating STAT6 in the MFP-2 knockdown cells, chromeceptin
exhibited little STAT6 activation even at 5 μM (Figure 1D).
411
Altogether, the results indicate that chromeceptin activates STAT6
through an MFP-2-dependent pathway that is different from the
pathway of IL-4 activation.
The chromeceptin–MFP-2 complex binds to ACC1 and impairs fatty
acid synthesis
To identify the protein factors that mediate chromeceptin-induced
signalling in STAT6 activation, MFP-2 and its associated proteins
were immunoprecipitated with an anti-MFP-2 antibody from
whole-cell extracts. Cells were treated with 0.1 % DMSO or
1 μM chromeceptin, and their extracts were immunoprecipitated
with an anti-MFP-2 antibody and Protein A beads. Masssequencing analysis of the precipitated protein bands in an
SDS/PAGE gel showed that the strong band at 80 kDa was
MFP-2 (Figure 2A), validating our immunopurification. The
sample from the chromeceptin-treated cells exhibited two clear
extra bands on an SDS gel. Mass-sequencing analysis of the bands
revealed their identities: ACC1 (UniProtKB/SwissProt accession
code Q13085) and PCCα (UniProtKB/SwissProt accession code
P05165). We did not detect any protein band for PCCβ, a binding
partner of PCCα. The apparent mass of the immunoprecipitated
ACC1 (≈120 kDa) was lower than that of full-length ACC1
(265 kDa). It remains unclear whether MFP-2 associates with
intact ACC1. The two low-molecular mass bands indicated by
stars (Figure 2A) were degraded fragments of MFP-2. It is
possible that ACC1, which is known to be highly susceptible
to proteolytic degradation [21,22], was degraded under our
purification conditions.
To confirm that ACC1 interacts with MFP-2 in the presence
of chromeceptin, we also performed co-IP experiments with an
anti-ACC1 antibody, in which proteins co-immunoprecipitated
with ACC1 were analysed using Western blotting with an
anti-MFP-2 antibody. The results supported our notion that
chromeceptin increases the association of ACC1 with MFP-2
(Figure 2B).
We examined the ability of chromeceptin to modulate the
enzymatic activity of these two carboxylases in cells. Wholecell extracts prepared from HepG2 cells treated with DMSO or
chromeceptin were incubated with acetyl-CoA or propionyl-CoA,
the substrates for ACC and PCC respectively. The quantities
of newly synthesized malonyl-CoA and methylmalonyl CoA
(products of ACC and PCC respectively) were monitored by
measuring the incorporation of a radioactive carbon source.
Production of malonyl-CoA was 50 % lower in the chromeceptintreated cells than in the DMSO-treated cells, whereas production
of methylmalonyl-CoA was similar (Figures 2C and 2D).
The selective reduction of malonyl-CoA synthesis in the
chromeceptin-treated cells suggests that chromeceptin impairs
ACC activity, but not PCC activity.
We next examined the effect of chromeceptin on de novo
fatty acid synthesis, where ACC1 catalyses the carboxylation of
acetyl-CoA to malonyl-CoA, a rate-limiting step in fatty acid
biosynthesis. HepG2 cells were treated with 1 μM chromeceptin
for 6 h, followed by a 12-h incubation with [14 C]acetate. The cells
were lysed, and saponifiable (i.e. fatty acids) and unsaponifiable
(i.e. cholesterol) lipids were quantified: there was 50 % decrease
of fatty acid synthesis, whereas cholesterol synthesis, which
doesn’t require ACC1, was unchanged (Figure 2E). These results
collectively suggest that chromeceptin inhibits ACC1 in cells.
We next examined the effects of chromeceptin on known acute
and chronic regulators of ACC1: protein stability, mRNA levels
[23] and phosphorylation [24,25]. Chromeceptin failed to alter
any of these at levels high enough to account for the 50 %
decrease of fatty acid synthesis (Supplementary Figures S1A
c The Authors Journal compilation c 2012 Biochemical Society
412
D. Jung and others
Figure 2 The chromeceptin–MFP-2 complex binds to ACC1 and impairs
fatty acid synthesis
(A) Isolation of ACC1. A Coomassie Blue G-250-stained SDS/PAGE gel of the proteins
co-immunoprecipitated with MFP-2 protein. In the presence of chromeceptin, ACC1 and PCCα
were co-immunoprecipitated with the MFP-2 protein. Stars indicate protein bands of degraded
fragments of MFP-2. IgG-H and IgG-L indicate bands of IgG heavy chain and light chain
respectively. (B) Chromeceptin increased the association of ACC1 with MFP-2. MFP-2 was
co-immunoprecipitated with ACC1 using an anti-ACC1 antibody in the presence or absence of
chromeceptin (1 μM). The electrophoretic mobilities of the bands are consistent with those
corresponding to full-length proteins. (C and D) Chromeceptin (1 μM) impairs the enzymatic
activity of ACCs (C), but has no significant effects on PCCs (D). HepG2 cells were treated
with 1 μM chromeceptin for 6 h, and the cell extracts were incubated with 0.4 mM acetyl-CoA
or propionyl-CoA for 5 min, to measure enzymatic activity of ACC or PCC respectively. The
amount of incorporated radiocarbon, from sodium [14 C]bicarbonate, into enzymatic products
of malonyl-CoA, by ACC, or methyl malonyl-CoA, by PCC, was measured. Error bars indicate
S.E.M. *P < 0.01, statistical significance using Student’s t test. (E) Chromeceptin (1 μM)
decreases de novo fatty acid synthesis, but has no significant effects on cholesterol synthesis.
HepG2 cells were treated with 1 μM chromeceptin for 6 h, followed by a 12-h incubation with
[14 C]acetate. The cells were lysed, and saponifiable (i.e. fatty acids) and unsaponifiable (i.e.
cholesterol) lipids were quantified. Error bars indicate S.E.M. *P < 0.01, statistical significance
using Student’s t test. Chrom, chromeceptin; DM, DMSO; M, molecular markers with masses
indicated in kDa; WB, Western blot.
and S1C at http://www.BiochemJ.org/bj/448/bj4480409add.htm).
Furthermore, there was no change in the protein levels of
MFP-2, PCCα and ACC2 in the presence of chromeceptin
(Supplementary Figure S1B). These observations implied that
the activity of ACC1 might be impaired by its interaction with
the chromeceptin–MFP-2 complex, which is different from the
known mechanisms for regulating ACC1 activity.
Chromeceptin induces translocation of ACC1 from the cytosol to
peroxisomes
ACC1 is a cytosolic protein, whereas MFP-2 is localized in
peroxisomes by its peroxisomal-targeting sequence. In order for
the two molecules to bind to each other, either ACC1 or MFP2
c The Authors Journal compilation c 2012 Biochemical Society
needs to be translocated. Immunohistochemical experiments
were carried out to visualize the localization of ACC1 and
MFP-2 in the presence or absence of chromeceptin. In cells
treated with DMSO alone, ACC1 appeared to be uniformly
distributed in the cytosol (Figure 3A), whereas MFP-2 was
localized in small areas (Figure 3B) that overlapped with those of
pmp70, a peroxisomal marker protein (Figure 3C). In contrast,
cells treated with chromeceptin exhibited speckles of ACC1
(Figure 3E) that overlapped with those of MFP-2 (Figure 3F)
and pmp70 (Figure 3G), indicating co-localization of ACC1 and
MFP-2 in peroxisomes (Figure 3H). Translocation of ACC1
was not detected upon IL-4 treatment (Supplementary Figure
S2 at http://www.BiochemJ.org/bj/448/bj4480409add.htm). Thus
MFP-2 remained in the peroxisomes, perhaps owing to its strong
peroxisomal-targeting sequence, whether or not cells were treated
with chromeceptin, and the chromeceptin-induced interaction
between MFP-2 and ACC1 appeared to occur in peroxisomes
through translocation of ACC1.
We next performed similar experiments with MFP-2 knockdown cells. Treatment of MFP-2 knockdown cells with 1 μM
chromeceptin for 1 h had little effect on the cytosolic localization
of ACC1 (compare Figures 4A and 4D), which had little overlap
with that of pmp70 (Figures 4C and 4F). These results provide
further evidence that ACC1 is translocated into peroxisomes
owing to its interaction with the chromeceptin–MFP-2
complex. Although a small amount of MFP-2 protein remained
in the knockdown cells, little translocation of ACC1 occurred,
suggesting that an abundance of MFP-2 protein might be required
to sequester ACC1 in peroxisomes.
To confirm the selective association of the chromeceptin–MFP2 complex with ACC1, we examined the effects of chromeceptin
on the subcellular localization of its isoform, ACC2. ACC2
is known to localize in mitochondria, due to its N-terminal
localization signal. As expected, cells treated with DMSO
alone exhibited mitochondrial localization of ACC2, which was
confirmed by overlapping images of an Alexa Fluor® 488labelled anti-ACC2 antibody and MitoTracker, a fluorescent dye
specific to mitochondria (Supplementary Figures S3A–S3C at
http://www.BiochemJ.org/bj/448/bj4480409add.htm). Treatment
with 1 μM chromeceptin for 1 h had no detectable effect on
the mitochondrial localization of ACC2 (Supplementary Figures
S3D–S3F). Localization of the other co-immunoprecipitated
protein, PCCα, was also examined in the presence or
absence of chromeceptin. Chromeceptin treatment failed to
induce clear translocation of PCCα into peroxisomes, or
colocalization with MFP-2 (Supplementary Figure S4 at
http://www.BiochemJ.org/bj/448/bj4480409add.htm). These results collectively suggest that ACC1 is selectively translocated
from the cytosol to peroxisomes by the chromeceptin–MFP2
complex.
Pex5p (PTS1 receptor)-dependent peroxisomal translocation of the
MFP-2–ACC1 complex
Peroxisomal translocation of PTS1-containing proteins is
guided by Pex5p, the peroxisomal cycling receptor [26,27].
Indeed, MFP-2, one of the PTS1-containing proteins, was
distributed in the cytosol in CHO-K1 cells that express
dysfunctional Pex5p (CHO-ZP105) (Supplementary Figure
S5F at http://www.BiochemJ.org/bj/448/bj4480409add.htm), a
G298E mutant of Pex5p which is devoid of its ability to transport
PTS1-containing proteins into peroxisomes [28], whereas
MFP-2 was localized in peroxisomes in wild-type CHOK1 cells (Supplementary Figure S5B and S5C). ACC1
Chromeceptin inhibits acetyl-CoA carboxylase 1
Figure 3
413
Chromeceptin-induced co-localization of ACC1 and MFP-2 in peroxisomes
HepG2 cells were treated with 0.1 % DMSO (A–D) or 1 μM chromeceptin (Chrom) (E–H) for 1 h, and ACC1 (A and E) and MFP-2 (B and F) were visualized with an Alexa Fluor® 488-labelled
anti-ACC1 antibody (green) and an Alexa Fluor® 647-labelled anti-MFP-2 antibody (blue) respectively. To indicate peroxisomes, pmp70 was visualized with an Alexa Fluor® 555-labelled anti-pmp70
antibody (red) (C and G). A/M/p indicates a superimposed fluorescent image of ACC1, MFP-2 and pmp70 (D and H). Scale bars, 10 μm.
induced translocation of ACC1 is dependent on the Pex5pdependent peroxisomal localization of MFP-2.
Malonyl-CoA is a mediator for chromeceptin-induced STAT6
activation
Figure 4
MFP-2-dependent peroxisomal translocation of ACC1
MFP-2 knockdown HepG2 cells were treated with 1 μM chromeceptin (Chrom) (A–C) or 0.1 %
DMSO (D–F) for 1 h. Neither DMSO nor chromeceptin treatment induced translocation of ACC1
(A and D) into peroxisomes where pmp70 is localized (B and E). ACC1 and pmp70 were
visualized as described in Figure 3. ACC1/pmp70 indicates a superimposed fluorescent image
of ACC1 (green) and pmp70 (red) (C and F). Scale bars, 10 μm.
remained in the cytosol in both wild-type CHO-K1 and
CHO-ZP105 in the absence of chromeceptin (Supplementary
Figures S5A and S5E). Treatment of cells with 1 μM
chromeceptin for 1 h translocated ACC1 into peroxisomes
(Figure 5A), where the peroxisome marker pmp70 (Figure 5B)
and MFP-2 (Figure 5C) are co-localized in wild-type
CHO-K1 cells. In contrast, chromeceptin treatment had no
detectable impact on the cytosolic localization of ACC1 in
CHO-ZP105 cells (Figure 5E), in which MFP-2 is distributed
in the cytosol (Figure 5G). Taken together with the results in
Figure 4, these results indicate that the observed chromeceptin-
Chromeceptin induces both STAT6 activation and translocation
of ACC1. To analyse the relationship between the two processes,
we examined the influence of siRNA knockdown of ACC1
on STAT6 activation. The siRNAs for ACC1 and the STAT6responsive reporter gene were transiently co-transfected into
HepG2 cells, and expression of the reporter gene was monitored.
Such co-transfection of multiple gene samples often increases the
population of the cells in which both genes are simultaneously
transfected [29]. Although HepG2 is known to be hard
to transiently transfect, co-transfection may permit detection of
the effects of siRNA on the reporter expression. The results
indeed showed that knockdown of ACC1 doubled expression of
the STAT6-responsive reporter gene compared with expression
in cells co-transfected with a control siRNA (Figure 6A). In
contrast, co-transfection of a plasmid encoding siPCCα failed
to activate the reporter gene. Repression levels of ACC1 and
PCCα expression are shown in Supplementary Figures S6(A)
and S6(B) (at http://www.BiochemJ.org/bj/448/bj4480409add.
htm). Despite the low transfection efficiency, siRNA-induced
repression of these two genes was detectable in Western blots.
These results suggest that inhibition of ACC1 is one mechanism
of chromeceptin-induced STAT6 activation.
Malonyl-CoA is the sole enzymatic product of ACC1,
suggesting that reduction of cellular malonyl-CoA levels might
activate STAT6. To reduce the levels of malonyl-CoA, the
STAT6 reporter gene was co-transfected into HepG2 cells with a
plasmid encoding MCD, an enzyme that converts malonly-CoA to
acetyl-CoA. Increased expression levels of MCD in transfected
cells were confirmed by Western blots (Supplementary Figure
S6C). Overexpression of MCD activated the reporter gene to an
extent similar to treatment with 1 μM chromeceptin (Figure 6B),
c The Authors Journal compilation c 2012 Biochemical Society
414
Figure 5
D. Jung and others
Pex5p-dependent peroxisomal translocation of ACC1
CHO-K1, a wild-type cell (A–D), and CHO-ZP105, a mutant cell which expresses inert Pex5p (E–H), were treated with 1 μM chromeceptin (Chrom) for 1 h. ACC1 (A and E), pmp70 (B and F) and
MFP-2 (C and G) were visualized as described in Figure 3. A/p/M indicates a superimposed fluorescent image of ACC1, pmp70 and MFP-2 (D and H). Scale bars, 10 μm.
Figure 6 Effects of ACC1 siRNA or overexpression of MCD on STAT6
activation
(A) Co-transfection of ACC1 siRNA and a STAT6 reporter gene showed activation of STAT6,
whereas co-transfection of PCCα siRNA failed to activate STAT6. (B) Co-transfection of a
plasmid encoding MCD and the STAT6 reporter gene showed activation of STAT6, which was
further enhanced by chromeceptin treatment compared with DMSO treatment alone. *P < 0.01,
statistically significant, calculated using Student’s t test. Error bars indicate S.E.M.
suggesting that down-regulation of cellular malonyl-CoA levels
activates STAT6.
DISCUSSION
Enzymatic activity of ACCs is regulated by several mechanisms
in response to changes in diet and hormones. Acute regulation
(minutes to hours) occurs through reversible phosphorylation
[24,25]; chronic regulation (hours to days) occurs through
alterations in mRNA stability and protein levels [23]. In
addition to these endogenous regulation mechanisms, a dozen
c The Authors Journal compilation c 2012 Biochemical Society
exogenous small molecules have been discovered or developed
that control the enzymatic activity of ACCs [30–33]. These
small molecules generally have an affinity for the enzymatic
domains of ACCs. For example, fatty acid mimetics, such
as aryloxyphenoxypropionate, cyclohexanedione herbicides and
substituted bipiperidylcarboxamide (e.g. CP-640186), inhibit
the enzymatic activity of the CT (carboxyltransferase) domain,
whereas soraphen A, a polyketide natural product, inhibits the
activity of the BC (biotin carboxylase) domain. These enzyme
inhibitors and their analogues show a limited preference for
a specific isoform, due to the high sequence similarity of the
enzymatic domains of ACC1 and ACC2. The amino acid sequence
similarities between ACC1 and ACC2 are 82 % and 76 %
respectively, for their BC and CT domains. Despite the high
sequence similarities, however, several small-molecule inhibitors
have recently been discovered to be selective for ACC2 [34]
or ACC1 [35]. The molecular mechanisms of their selectivities
remain unknown. The present study suggests that chromeceptin
selectively impairs ACC1 by inducing the interaction between
ACC1 and MFP-2. The basis of the selective association of ACC1
with the chromeceptin–MFP-2 complex remains unclear. The
tight anchoring of ACC2 to mitochondria may render it
unavailable for interaction with the chromeceptin–MFP-2
complex and thereby for translocation into peroxisomes. Another
explanation would be that the ACC1 selectivity is achieved by a
broad and selective binding surface of the chromeceptin–MFP-2
complex.
The ‘dimerizer’ activity of chromeceptin is similar to inhibition
of calcineurin by FK506, an immunosuppressive natural product.
FK506 blocks the protein phosphatase activity of calcineurin
in immunosuppression by mediating the interaction between
calcineurin and FKBPs (FK506-binding proteins) [36]. Similarly,
chromeceptin appears to block the enzymatic activity of ACC1
by mediating the interaction between ACC1 and MFP-2. The
ability of chromeceptin to inhibit ACC activity even in cell lysates
(Figure 2C) supports such a direct inhibition mechanism.
In the case of FK506, however, both calcineurin and FKBPs are
cytosolic proteins. In contrast, ACC1 and MFP-2 are localized
in different cellular components. It remains unclear how these
three components meet together in cells. One possibility is that
Chromeceptin inhibits acetyl-CoA carboxylase 1
ACC1 might interact with the chromeceptin–MFP-2 complex
in the cytosol, then translocates into peroxisomes through the
guidance of Pex5p. The peroxisomal import machinery allows
the penetration of folded proteins, oligomerized proteins and
even gold nanoparticles that have peroxisomal-targeting signals
[37,38]. Proteins that lack PTS1 can also be transported, when
they are bound to a PTS1-containing partner protein [39],
suggesting that the peroxisomal import machinery can transport
a large protein complex such as the chromeceptin–MFP-2–ACC1
complex. Another possibility is that peroxisomal ACC1 interacts
with the chromeceptin–MFP-2 complex in the peroxisome and
accumulates there. Further studies are needed for detailed
mechanisms of chromeceptin-induced translocation of ACC1.
Translocation of ACC1 suggests an additional mechanism
of enzyme inhibition: sequestration. ACC1 sequestered in
peroxisomes is unlikely to contribute to malonyl-CoA levels in
the cytosol, where fatty acids are synthesized from malonylCoA and acetyl-CoA, because malonyl-CoA in peroxisomes is
disposed of by MCD [40]. This notion is consistent with the
significant decrease in de novo fatty acid synthesis from acetate
in chromeceptin-treated HepG2 cells. ACC1 sequestration may
also disrupt metabolons in cytosol. It has been proposed that
metabolic enzymes, including ACC1, are organized in these
large protein complexes. The activities of highly purified ACCs
are not adequate to account for enzyme function in intact cells
[41], and their occurrence in metabolons might be necessary to
optimize function. Association of the ACC1 homologues with
other proteins, including a protein phosphatase and a heat-shock
protein, has been described in Drosophila melanogaster [42] and
Caenorhabditis elegans [43]. A 75 kDa regulatory protein that
activates ACC has been co-purified with ACC from rat liver;
blocking the interaction between this protein and ACC reduces
the enzymatic activity of ACC [41]. Disruption of large ACC1containing protein complexes by chromeceptin may impair the
ability of ACC1 to synthesize malonyl-CoA.
No direct functional association between MFP-2 and ACC1
has been previously reported, even though both are involved in
fatty acid metabolism, and MFP-2 is a multi-functional protein
that is essential for the maturation of bile acids and peroxisomal
β-oxidation [15,17]. It is possible that chromeceptin mimics an
endogenous fatty acid-related ligand that induces the interaction
of MFP-2 and ACC1 to control ACC1 activity. Search for such
an endogenous ligand is in progress.
ACC1 has been proposed as a target drug for cancer therapy,
because it supplies precursors for the de novo synthesis of fatty
acids that are required for the growth and survival of cancer
cells. Knockdown of ACC1 results in growth inhibition and
apoptosis of prostate cancer cells [44], and ACC1 is essential
for the survival of breast cancer cells [45]. The molecular
mechanism of chromeceptin may provide new pharmacological
approaches to selective inhibition of ACC1 for biological studies
and pharmaceutical development.
AUTHOR CONTRIBUTION
Dongju Jung and Motonari Uesugi designed the study. Dongju Jung performed all of
the experiments with support from Lutfi Abu-Elheiga, Rie Ayuzawa, Ziwei Gu, Takashi
Shirakawa, Yukio Fujiki, Norio Nakatsuji and Salih Wakil. Dongju Jung and Motonari
Uesugi wrote the paper.
FUNDING
This work was supported in part by the JSPS (Japan Society for the Promotion of Science)
[grant-in-aid 21310140 and LR018 (to M.U.)], the Hoh-ansha Foundation (to M.U.),
the Hefni Technical Training Foundation (to S.J.W.), the Takeda Science Foundation (to
415
D.J.) and the National Institutes of Health [grant number GM-63115 (to S.J.W.)]. The
Kyoto research group is supported by the World Premier International Research Center
Initiative (WPI), MEXT (Ministry of Education, Culture, Sports, Science and Technology),
Japan, and participates in the Global COE program ‘Integrated Materials Science’
(#B-09).
REFERENCES
1 Wakil, S. J., Titchener, E. B. and Gibson, D. M. (1958) Evidence for the participation of
biotin in the enzymic synthesis of fatty acids. Biochim. Biophys. Acta 29, 225–226
2 Wakil, S. J. (1962) Lipid metabolism. Annu. Rev. Biochem. 31, 369–406
3 Thampy, K. G. (1989) Formation of malonyl coenzyme A in rat heart. Identification and
purification of an isozyme of A carboxylase from rat heart. J. Biol. Chem. 264,
17631–17634
4 Ha, J., Daniel, S., Kong, I. S., Park, C. K., Tae, H. J. and Kim, K. H. (1994) Cloning of
human acetyl-CoA carboxylase cDNA. Eur. J. Biochem. 219, 297–306
5 Abu-Elheiga, L., Jayakumar, A., Baldini, A., Chirala, S. S. and Wakil, S. J. (1995) Human
acetyl-CoA carboxylase: characterization, molecular cloning, and evidence for two
isoforms. Proc. Natl. Acad. Sci. U.S.A. 92, 4011–4015
6 Abu-Elheiga, L., Almarza-Ortega, D. B., Baldini, A. and Wakil, S. J. (1997) Human
acetyl-CoA carboxylase 2. Molecular cloning, characterization, chromosomal mapping,
and evidence for two isoforms. J. Biol. Chem. 272, 10669–10677
7 Abu-Elheiga, L., Brinkley, W. R., Zhong, L., Chirala, S. S., Woldegiorgis, G. and Wakil,
S. J. (2000) The subcellular localization of acetyl-CoA carboxylase 2. Proc. Natl. Acad.
Sci. U.S.A. 97, 1444–1449
8 Abu-Elheiga, L., Matzuk, M. M., Kordari, P., Oh, W., Shaikenov, T., Gu, Z. and Wakil, S. J.
(2005) Mutant mice lacking acetyl-CoA carboxylase 1 are embryonically lethal. Proc.
Natl. Acad. Sci. U.S.A. 102, 12011–12016
9 McGarry, J. D. and Brown, N. F. (1997) The mitochondrial carnitine palmitoyltransferase
system. From concept to molecular analysis. Eur. J. Biochem. 244, 1–14
10 Abu-Elheiga, L., Matzuk, M. M., Abo-Hashema, K. A. and Wakil, S. J. (2001) Continuous
fatty acid oxidation and reduced fat storage in mice lacking acetyl-CoA carboxylase 2.
Science 291, 2613–2616
11 Harwood, Jr, H. J., Petras, S. F., Shelly, L. D., Zaccaro, L. M., Perry, D. A., Makowski,
M. R., Hargrove, D. M., Martin, K. A., Tracey, W. R., Chapman, J. G. et al. (2003)
Isozyme-nonselective N-substituted bipiperidylcarboxamide acetyl-CoA carboxylase
inhibitors reduce tissue malonyl-CoA concentrations, inhibit fatty acid synthesis, and
increase fatty acid oxidation in cultured cells and in experimental animals. J. Biol. Chem.
278, 37099–37111
12 Beckers, A., Organe, S., Timmermans, L., Scheys, K., Peeters, A., Brusselmans, K.,
Verhoeven, G. and Swinnen, J. V. (2007) Chemical inhibition of acetyl-CoA carboxylase
induces growth arrest and cytotoxicity selectively in cancer cells. Cancer Res. 67,
8180–8187
13 Choi, Y., Kawazoe, Y., Murakami, K., Misawa, H. and Uesugi, M. (2003) Identification of
bioactive molecules by adipogenesis profiling of organic compounds. J. Biol. Chem.
278, 7320–7324
14 Choi, Y., Shimogawa, H., Murakami, K., Ramdas, L., Zhang, W., Qin, J. and Uesugi, M.
(2006) Chemical genetic identification of the IGF-linked pathway that is mediated by
STAT6 and MFP2. Chem. Biol. 13, 241–249
15 Huyghe, S., Mannaerts, G. P., Baes, M. and Van Veldhoven, P. P. (2006) Peroxisomal
multifunctional protein-2: the enzyme, the patients and the knockout mouse model.
Biochim. Biophys. Acta 1761, 973–994
16 Moller, G., Luders, J., Markus, M., Husen, B., Van Veldhoven, P. P. and Adamski, J.
(1999) Peroxisome targeting of porcine 17β-hydroxysteroid dehydrogenase
type IV/D-specific multifunctional protein 2 is mediated by its C-terminal tripeptide AKI.
J. Cell. Biochem. 73, 70–78
17 Baes, M., Huyghe, S., Carmeliet, P., Declercq, P. E., Collen, D., Mannaerts, G. P. and Van
Veldhoven, P. P. (2000) Inactivation of the peroxisomal multifunctional protein-2 in mice
impedes the degradation of not only 2-methyl-branched fatty acids and bile acid
intermediates but also of very long chain fatty acids. J. Biol. Chem. 275,
16329–16336
18 Thampy, K. G. and Wakil, S. J. (1985) Activation of acetyl-CoA carboxylase.
Purification and properties of a Mn2 + -dependent phosphatase. J. Biol. Chem. 260,
6318–6323
19 Mao, J., DeMayo, F. J., Li, H., Abu-Elheiga, L., Gu, Z., Shaikenov, T. E., Kordari, P.,
Chirala, S. S., Heird, W. C. and Wakil, S. J. (2006) Liver-specific deletion of acetyl-CoA
carboxylase 1 reduces hepatic triglyceride accumulation without affecting glucose
homeostasis. Proc. Natl. Acad. Sci. U.S.A. 103, 8552–8557
20 Mann, M., Hendrickson, R. C. and Pandey, A. (2001) Analysis of proteins and proteomes
by mass spectrometry. Annu. Rev. Biochem. 70, 437–473
c The Authors Journal compilation c 2012 Biochemical Society
416
D. Jung and others
21 Tanabe, T., Wada, K., Okazaki, T. and Numa, S. (1975) Acetyl-coenzyme-A carboxylase
from rat liver. Subunit structure and proteolytic modification. Eur. J. Biochem. 57,
15–24
22 Wada, K. and Tanabe, T. (1985) Proteolysis of liver acetyl coenzyme A carboxylase by
cathepsin B. FEBS Lett. 180, 74–76
23 Pape, M. E. and Kim, K. H. (1989) Transcriptional regulation of acetyl coenzyme A
carboxylase gene expression by tumor necrosis factor in 30A-5 preadipocytes. Mol. Cell.
Biol. 9, 974–982
24 Kim, K. H., Lopez-Casillas, F., Bai, D. H., Luo, X. and Pape, M. E. (1989) Role of
reversible phosphorylation of acetyl-CoA carboxylase in long-chain fatty acid synthesis.
FASEB J. 3, 2250–2256
25 Ha, J., Daniel, S., Broyles, S. S. and Kim, K. H. (1994) Critical phosphorylation sites for
acetyl-CoA carboxylase activity. J. Biol. Chem. 269, 22162–22168
26 Dodt, G., Braverman, N., Wong, C., Moser, A., Moser, H. W., Watkins, P., Valle, D. and
Gould, S. J. (1995) Mutations in the PTS1 receptor gene, PXR1, define complementation
group 2 of the peroxisome biogenesis disorders. Nat. Genet. 9, 115–125
27 Rachubinski, R. A. and Subramani, S. (1995) How proteins penetrate peroxisomes. Cell
83, 525–528
28 Otera, H., Okumoto, K., Tateishi, K., Ikoma, Y., Matsuda, E., Nishimura, M., Tsukamoto, T.,
Osumi, T., Ohashi, K., Higuchi, O. and Fujiki, Y. (1998) Peroxisome targeting signal type 1
(PTS1) receptor is involved in import of both PTS1 and PTS2: studies with
PEX5-defective CHO cell mutants. Mol. Cell. Biol. 18, 388–399
29 Yamano, S., Dai, J. and Moursi, A. M. (2010) Comparison of transfection efficiency of
nonviral gene transfer reagents. Mol. Biotechnol. 46, 287–300
30 Harwood, Jr, H. J. (2005) Treating the metabolic syndrome: acetyl-CoA carboxylase
inhibition. Expert. Opin. Ther. Targets 9, 267–281
31 Tong, L. and Harwood, Jr, H. J. (2006) Acetyl-coenzyme A carboxylases: versatile targets
for drug discovery. J. Cell. Biochem. 99, 1476–1488
32 Tong, L. (2005) Acetyl-coenzyme A carboxylase: crucial metabolic enzyme and attractive
target for drug discovery. Cell. Mol. Life Sci. 62, 1784–1803
33 Shen, Y., Volrath, S. L., Weatherly, S. C., Elich, T. D. and Tong, L. (2004) A mechanism for
the potent inhibition of eukaryotic acetyl-coenzyme A carboxylase by soraphen A, a
macrocyclic polyketide natural product. Mol. Cell 16, 881–891
Received 23 July 2012/8 October 2012; accepted 15 October 2012
Published as BJ Immediate Publication 15 October 2012, doi:10.1042/BJ20121158
c The Authors Journal compilation c 2012 Biochemical Society
34 Gu, Y. G., Weitzberg, M., Clark, R. F., Xu, X., Li, Q., Zhang, T., Hansen, T. M., Liu, G., Xin,
Z., Wang, X. et al. (2006) Synthesis and structure-activity relationships of
N-{3-[2-(4-alkoxyphenoxy)thiazol-5-yl]-1- methylprop-2-ynyl}carboxy derivatives as
selective acetyl-CoA carboxylase 2 inhibitors. J. Med. Chem. 49, 3770–3773
35 Haque, T. S., Liang, N., Golla, R., Seethala, R., Ma, Z., Ewing, W. R., Cooper, C. B.,
Pelleymounter, M. A., Poss, M. A. and Cheng, D. (2009) Potent biphenyl- and 3-phenyl
pyridine-based inhibitors of acetyl-CoA carboxylase. Bioorg. Med. Chem. Lett. 19,
5872–5876
36 Liu, J., Farmer, Jr, J. D., Lane, W. S., Friedman, J., Weissman, I. and Schreiber, S. L.
(1991) Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506
complexes. Cell 66, 807–815
37 Schnell, D. J. and Hebert, D. N. (2003) Protein translocons: multifunctional mediators of
protein translocation across membranes. Cell 112, 491–505
38 Walton, P. A., Hill, P. E. and Subramani, S. (1995) Import of stably folded proteins into
peroxisomes. Mol. Biol. Cell 6, 675–683
39 Glover, J. R., Andrews, D. W. and Rachubinski, R. A. (1994) Saccharomyces cerevisiae
peroxisomal thiolase is imported as a dimer. Proc. Natl. Acad. Sci. U.S.A. 91,
10541–10545
40 Saggerson, D. (2008) Malonyl-CoA, a key signaling molecule in mammalian cells. Annu.
Rev. Nutr. 28, 253–272
41 Quayle, K. A., Denton, R. M. and Brownsey, R. W. (1993) Evidence for a protein regulator
from rat liver which activates acetyl-CoA carboxylase. Biochem. J. 292, 75–84
42 Giot, L., Bader, J. S., Brouwer, C., Chaudhuri, A., Kuang, B., Li, Y., Hao, Y. L., Ooi, C. E.,
Godwin, B., Vitols, E. et al. (2003) A protein interaction map of Drosophila melanogaster .
Science 302, 1727–1736
43 Li, S., Armstrong, C. M., Bertin, N., Ge, H., Milstein, S., Boxem, M., Vidalain, P. O., Han,
J. D., Chesneau, A., Hao, T. et al. (2004) A map of the interactome network of the
metazoan C. elegans . Science 303, 540–543
44 Brusselmans, K., De Schrijver, E., Verhoeven, G. and Swinnen, J. V. (2005) RNA
interference-mediated silencing of the acetyl-CoA-carboxylase-α gene induces growth
inhibition and apoptosis of prostate cancer cells. Cancer Res. 65, 6719–6725
45 Chajes, V., Cambot, M., Moreau, K., Lenoir, G. M. and Joulin, V. (2006) Acetyl-CoA
carboxylase α is essential to breast cancer cell survival. Cancer Res. 66, 5287–5294
Biochem. J. (2012) 448, 409–416 (Printed in Great Britain)
doi:10.1042/BJ20121158
SUPPLEMENTARY ONLINE DATA
Mislocalization and inhibition of acetyl-CoA carboxylase 1 by a synthetic
small molecule
Dongju JUNG*1 , Lutfi ABU-ELHEIGA†, Rie AYUZAWA*, Ziwei GU†, Takashi SHIRAKAWA‡, Yukio FUJIKI§, Norio NAKATSUJI*,
Salih J. WAKIL† and Motonari UESUGI*‡1
*Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto 606-8501, Japan, †Verna and Marrs McLean Department of Biochemistry and Molecular Biology,
Baylor College of Medicine, Houston, TX 77030, U.S.A., ‡Institute for Chemical Research, Kyoto University, Uji 611-0011, Japan, and §Department of Biology, Kyushu University
Faculty of Science, Fukuoka 812-81, Japan
Figure S1
Examination of protein and mRNA levels
For Western blot analysis, HepG2 cells were treated with chromeceptin for 6 h under serum-free
conditions. To check the protein levels, 10 μg from each extract were used. (A) Protein expression
and phosphorylation status of ACC1 (Ser79 ) monitored by Western blotting. Anti-ACC antibodies
were from U.S. Biological and the anti-phophospho-ACC1 (Ser79 ) antibody was from Cell
Signaling. DM indicates 0.1 % DMSO (vehicle) alone. Concentrations of chromeceptin were
0–4 μM. (B) PCCα, MFP-2 and ACC2 expression levels monitored by Western bloting.
Anti-propionyl-CoA carboxylase antibody was from Abcam, and its secondary anti-chicken
IgY horseradish-peroxidase-conjugated antibody was from Millipore. Anti-MFP-2 antibody was
raised in rabbit against the 15 C-terminal amino acids (CSQKLQMILKDYAKL) of human MFP-2
protein (NCBI Reference Sequence NP_000405.1). Anti-ACC2 antibody was from US Biological.
DM indicates 0.1 % DMSO (vehicle) alone. Concentrations of chromeceptin were 0–5 μM. (C)
To quantify ACC1 mRNA levels, 1 μg of total RNA was extracted with RNeasy Mini kit (Qiagen)
and used to synthesize first-strand cDNA with ReverTra Ace (Toyobo). Real-time PCR was
performed using SYBR® Green Master mix (Toyobo) with an Applied Biosystem 7300. Primers
were designed for ACC1 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase), on the
basis of the sequence information in PrimerBank (http://pga.mgh.harvard.edu/primerbank/).
The expression of ACC1 was normalized to the expression of GAPDH. All reactions were
performed in triplicate.
1
Figure S2 Treatment with chromeceptin, but not IL-4, results in localization
of ACC1 in peroxisomes
HepG2 cells were treated with 0.1 % DMSO (A–C), 50 ng/ml IL-4 (D–F) or 1 μM chromeceptin
(Chrom) (G–I) for 1 h, then fixed. ACC1 and MFP-2 were visualized as described in Figure 3
of the main text. ACC1 was distributed in cytosol (A and D), and MFP-2 was localized in
peroxisome (B and E) in cells treated with DMSO or IL-4. Chromeceptin treatment induced
translocation of ACC1 into peroxisomes (G) where MFP-2 localized (H). (C), (F) and (I) show
merged images of ACC1 and MFP-2. Scale bars, 10 μm.
Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
D. Jung and others
Figure S3
Stationary localization of ACC2 in mitochondria
HepG2 cells were treated with 0.1 % DMSO (A–C) or 1 μM chromeceptin (Chrom) (D–F) for
1 h. ACC2 and mitochondria were visualized with an Alexa Fluor® 488-labelled anti-ACC2
antibody (green) and MitoTracker dye® (Mito; red) respectively. Staining of ACC2 showed
characteristic mitochondrial localization both in DMSO- and chromeceptin-treated cells (A and
D), similar to those of MitoTracker (B and E). (C) and (F) show merged images. Scale bars,
10 μm.
Figure S4
Chromeceptin has little effect on translocation of PCCα
HepG2 cells were treated with 0.1 % DMSO (A–C) or 1 μM chromeceptin (Chrom) (D–F) for 1 h,
and then fixed. PCCα was visualized with an Alexa Fluor® 488-labelled anti-PCCα antibody
(green) (A and D) and MFP-2 was visualized with an Alexa Fluor® 555-labelled anti-MFP-2
antibody (red) (B and E). Unlike ACC1, PCCα remained in the cytosol even after chromeceptin
treatment (compare A and D). MFP-2 localized in peroxisomes (B and E), showing little overlap
with PCCα (C and F). Scale bars, 10 μm.
c The Authors Journal compilation c 2012 Biochemical Society
Chromeceptin inhibits acetyl-CoA carboxylase 1
Figure S5
Impairment of peroxisomal translocation of ACC1 and MFP-2 in a Pex5p mutant cell
CHO-K1 (wild-type) and CHO-ZP105 (Pex5p mutant) cells were treated with 0.1 % DMSO for 1 h. ACC1 (green; A and E), pmp70 (red; C and G) and MFP-2 (blue; B and F) were visualized with
fluorescence-labelled antibodies as described in Figure 3 of the main text. ACC1 was distributed in the cytosol both in wild-type (A) and mutant cells (E), whereas MFP-2 localized in the peroxisome
in CHO-K1 cells (B) and in the cytosol in CHO-ZP105 cells (F). Pmp70 localized in peroxisomes regardless of the Pex5p mutation (C and G). (D) and (H) show merged images. Scale bars, 10 μm.
Figure S6
of MCD
Repression of ACC1 and PCCα with siRNA, and overexpression
HepG2 cells were transiently transfected with an siRNA for ACC1, and a plasmid encoding MCD
or siRNA for PCCα. Expression level of ACC1 (A), PCCα (B) and MCD (C) were analysed by
Western bloting 24 h later. Loading control for each protein was indicated by actin level. siCon
and Con indicate transfection of non-specific siRNA and empty plasmid respectively.
Received 23 July 2012/8 October 2012; accepted 15 October 2012
Published as BJ Immediate Publication 15 October 2012, doi:10.1042/BJ20121158
c The Authors Journal compilation c 2012 Biochemical Society