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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.)]. 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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