Peptides from purified soybean b-conglycinin inhibit fatty acid synthase by interaction with the thioesterase catalytic domain Cristina Martinez-Villaluenga1, Sanjeewa G Rupasinghe2, Mary A Schuler2 and Elvira Gonzalez de Mejia1 Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, IL, USA Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, IL, USA Keywords b-conglycinin-derived peptides; fatty acid synthase; inhibitors; soybean; thioesterase Correspondence E Gonzalez de Mejia, 1201 West Gregory Drive, 228 ERML, MC-051, Urbana, IL 61801, USA Fax: +1 217 265 0925 Tel: +1 217 244 3196 E-mail: edemejia@illinois.edu (Received 19 August 2009, revised January 2010, accepted January 2010) doi:10.1111/j.1742-4658.2010.07577.x Fatty acid synthase (FAS) is uniquely expressed at high levels in cancer cells and adipose tissue The objectives of this study were to identify, purify and validate soy FAS inhibitory peptides and to predict their binding modes Soy peptides were isolated from hydrolysates of purified b-conglycinin by co-immunoprecipitation and identified using LC-MS ⁄ MS Three peptides, KNPQLR, EITPEKNPQLR and RKQEEDEDEEQQRE, inhibited FAS The biological activity of these peptides was confirmed by their inhibitory activity against purified chicken FAS (IC50 = 79, 27 and 16 lm, respectively) and a high correlation (r = )0.7) with lipid accumulation in 3T3-L1 adipocytes The FAS inhibitory potency of soy peptides also correlated with their molecular mass, pI value and the number of negatively charged and hydrophilic residues Molecular modeling predicted that the large FAS inhibitory peptides (EITPEKNPQLR and RKQEEDEDEEQQRE) bond to the thioesterase domain of human FAS with lower interaction energies ()442 and )353 kcalỈmol)1, respectively) than classical thioesterase inhibitors (Orlistat, )91 kcalỈmol)1 and C75, )51 kcalỈmol)1) Docking studies suggested that soy peptides blocked the active site through interactions within the catalytic triad, the interface cavity and the hydrophobic groove in the human FAS thioesterase domain FAS thioesterase inhibitory activities displayed by the synthetic soy peptides EITPEKNPQLR and RKQEEDEDEEQQRE (IC50 = 10.1 ± 1.6 and 10.7 ± 4.4 lm, respectively) were higher than C75 (58.7 lm) but lower than Orlistat (0.9 lm) This is the first study to identify FAS inhibitory peptides from purified b-conglycinin hydrolysates and predict their binding modes at the molecular level, leading to their possible use as nutraceuticals Structured digital abstract l MINT-7544766, MINT-7546418, MINT-7546830: Beta-conglycinin (uniprotkb:P25974) binds (MI:0407) to Alpha subunit of BC (uniprotkb:P13916) by pull down (MI:0096) l MINT-7547140, MINT-7547249: Beta-conglycinin (uniprotkb:P25974) binds (MI:0407) to Beta subunit of BC (uniprotkb:P25974) by pull down (MI:0096) Abbreviations ACP, acyl carrier protein; CIP, co-immunoprecipitation; DMEM, Dulbecco’s modified Eagle’s medium; ER, b-enoyl reductase; FAS, fatty acid synthase; PDB, Protein Data Bank; SBC, soybean b-conglycinin; TBST, Tris-buffered saline containing 0.1% Tween 20; TE, thioesterase FEBS Journal 277 (2010) 1481–1493 ª 2010 The Authors Journal compilation ª 2010 FEBS 1481 b-conglycinin peptides inhibit fatty acid synthase C Martinez-Villaluenga et al Introduction Fatty acid synthase (FAS, EC 3.2.1.85) is a multicomponent enzyme that catalyzes the de novo biosynthesis of long-chain fatty acids from acetyl-CoA and malonyl-CoA through a NADPH-dependent cyclic reaction [1] FAS is homodimeric and each polypeptide chain (270 kDa) carries seven catalytic domains integrating all the steps needed for fatty acid synthesis [2,3] The growing fatty acid is covalently attached to an acyl carrier protein (ACP), which transports it through the active sites where each reaction is catalyzed Once the fatty acid reaches 16–18 carbon atoms in length, it is released by the thioestherase (TE) domain [1] Human FAS is downregulated in most normal human tissues but is highly expressed in adipose and malignant tissues [4,5] Because of FAS overexpression in certain chronic diseases, it has become an important molecular target for chemoprevention and therapeutic intervention [6,7] The discovery and development of pharmacologic FAS inhibitors promise the prevention of obesity, related metabolic disorders and cancer [5,8] Inhibition of FAS in the central nervous system markedly reduces food intake and body weight in animal models [9] In particular, inhibition of FAS in the hypothalamus and pancreatic b cells protects mice against high fat diet-induced metabolic syndrome [10] Pharmacological inhibition of FAS results in a 7–10% longer survival time in mice with gastrointestinal cancer [11] Cerulenin and C75 represent two synthetic compounds reported to inhibit FAS [12] but their use has been limited by several drawbacks, including their irreversible behavior, low specificity, high chemical reactivity, interference with other cellular processes and controversial toxic effects [13–15] Orlistat (tetrahydrolipstatin) is an anti-obesity drug intended to inhibit gastric and pancreatic lipases [16] but it has been found to inhibit FAS by interacting with its TE domain; however, it has shown poor systemic stability and bioavailability [17] The selective FAS inhibitor GSK837149A discovered by Vazquez et al [18] was shown to have very low cell permeability in cell cul- ture Additional plant-derived compounds have also been discovered as potential FAS inhibitors [19–22] Our previous in vitro studies have shown that soybean b-conglycinin (SBC) contains active peptides that inhibit FAS [23] and fatty acid biosynthesis in adipocytes [24] The objectives of this study were to identify SBC-derived peptides with FAS inhibitory activity using co-immunoprecipitation (CIP) and proteomic techniques The FAS inhibitory activity of the identified peptides was established in both biochemical assays and cell-based models of 3T3-L1 adipocytes The relationship between the chemical characteristics of these peptides and their FAS inhibitory potency was defined, and their binding modes were predicted by docking simulations using the crystal structures of mammalian FAS (PBD ID code: 2VZ8) [25] and human FAS (PBD ID code: 2PX6) [17] This study provides valuable information for the rational design of new FAS inhibitors and the preparation of natural compounds potentially preventing the development of cancer, obesity and related metabolic disorders Results Identification of FAS inhibitory peptides from purified SBC hydrolysate Previous work in our laboratory demonstrated that hydrolysates from b-conglycinin with alcalase (Bacillus licheniformis) exert a potent inhibitory effect (IC50 = 30 lm) on FAS activity [23] In this study, amino acid sequences, identified by LC-MS ⁄ MS, of FAS inhibitory peptides co-immunoprecipitated from purified SBC hydrolysate were found to be KNPQLR (758 Da), EITPEKNPQLR (1324 Da) and RKQEEDEDEEQQRE (1847 Da) (Table 1) blast results indicated that the peptides KNPQLR and EITPEKNPQLR matched sequences present in both the a and b subunits of SBC, whereas RKQEEDEDEEQQRE matched sequences in the a subunit Table Identification of co-immunoprecipitated soybean b-conglycinin-derived peptides by LC-MS ⁄ MS The peptide sequences listed were found with a confidence of at least 95% (P < 0.05) Experimental mass (Da) Theoretical mass (Da) Putative sequence Protein fragment Protein source 754.18 754.18 1323.79 1323.79 1846.88 754.44 754.44 1323.72 1323.72 1846.79 KNPQLR KNPQLR EITPEKNPQLR EITPEKNPQLR RKQEEDEDEEQQRE f f f f f a b a b a 1482 (428–434) (263–268) (424–434) (258–268) (165–178) subunit subunit subunit subunit subunit of of of of of b-conglycinin b-conglycinin b-conglycinin b-conglycinin b-conglycinin Accession no P13916 P25974 P13916 P25974 P13916 FEBS Journal 277 (2010) 1481–1493 ª 2010 The Authors Journal compilation ª 2010 FEBS C Martinez-Villaluenga et al b-conglycinin peptides inhibit fatty acid synthase 120 FAS activity inhibition (%) KNPQLR EITPEKNPQLR 100 a RKQEEDEDEEQQRE C75 80 abc bcd 60 40 20 def def defg fg f 12 a a 25 cde efg 30 50 [Compound] (µM) 60 150 Fig Fatty acid synthase (FAS) inhibitory activity of synthetic peptides and C75 Synthetic peptides KNPQLR, EITPEKNPQLR and RKQEEDEDEEQQRE inhibited FAS in a dose-dependent manner which was similar to the positive control C75 Evaluation of FAS activity was performed after 20-min preincubation with different concentrations of each compound Values were expressed as percent inhibition of FAS activity compared with a negative control that included no inhibitors Each dataset corresponds to the mean of three independent replicates with error bars indicating the standard deviations Different letters indicate significant differences at P < 0.05 in one-way ANOVA analysis Confirmation of the FAS inhibitory activity of the identified peptides To confirm that the identified peptides (KNPQLR, EITPEKNPQLR and RKQEEDEDEEQQRE) inhibited FAS activity, their amino acid sequences were custom synthesized and their FAS inhibitory activities tested Figure shows the FAS inhibitory activity of the three synthetic peptides and the C75 positive control The FAS inhibitory responses for each of these peptides were dose dependent, reaching 38.2, 76.5 and 79.4% inhibition at 50 lm for KNPQLR, EITPEKNPQLR and RKQEEDEDEEQQRE, respectively compared with 77.8% inhibition at 150 lm for C75 Interestingly, although the three synthetic peptides showed FAS inhibitory activity, their potency was different RKQEEDEDEEQQRE exerted a strong inhibitory activity ($ 40% inhibition) at doses as low as 12 lm To compare the potency of the FAS inhibitory response across all of the peptides and the C75 positive control, classical sigmoidal dose–response curves were plotted and used to calculate the IC50 values listed in Table In these, the larger peptides, RKQEEDEDEEQQRE (IC50 = 16.5 lm) and EITPEKNPQLR (IC50 = 27.4 lm), showed significantly higher (P < 0.05) potency (lower IC50 value) than the C75 positive control (IC50 = 80.3 lm) The smaller KNPQLR peptide had a higher IC50 value (79.9 lm) (P < 0.05) more comparable with the positive control C75 (P > 0.05) Structure–potency relationship of FAS inhibitory peptides To further understand the structure–potency relationship of the FAS inhibitory peptides, we examined the relationships between the physicochemical and biochemical features of these peptides and their respective inhibitory potency (Table 2) Positive correlations were observed between their potency (lower IC50 value, Table Fatty acid synthase (FAS) inhibitory potency and physicochemical and biochemical characteristics of synthetic peptides MM, peptide molecular mass; pI, theoretical isoelectric point of each peptide; GRAVY, grand average of hydropathicity index Parameters were obtained using the Protparam tool in the ExPASY Proteomic Server Physicochemical properties No of charged residues Biological activity IC50 value of FAS inhibitory activity (lM)a KNPQLR EITPEKNPQLR RKQEEDEDEEQQRE C75 (positive control) Correlation coefficient (r)d a 79.9 27.4 16.5 80.3 ± ± ± ± 15.3b 7.8c 2.9c 19.5b MM pI Negative Positive Hydrophilic amino acids (%) 754.8 1324.5 1847.8 254.3 +0.89 11 6.2 4.3 – +0.64 2 – +0.40 13 – +0.69 )0.99 GRAVY Aliphatic index )2.2 )1.63 )3.67 – )0.16 65.0 70.9 – )0.33 b,c Data represent the mean ± SD of three independent experiments; Different letters (b,c) in the column indicate statistical difference (P < 0.05, in one-way ANOVA analysis) d Statistical correlations were carried out between the indicated parameter with the FAS inhibitory potency (the lower the IC50 value the higher the potency) of each peptide FEBS Journal 277 (2010) 1481–1493 ª 2010 The Authors Journal compilation ª 2010 FEBS 1483 b-conglycinin peptides inhibit fatty acid synthase C Martinez-Villaluenga et al MAT KS ACP DH ER KR TE higher potency), their molecular masses (r = +0.89) and the number of negatively charged (r = +0.64) and hydrophilic (r = +0.69) residues By contrast, a strong negative correlation was observed between their potency and their pI values (r = )0.99) Together, these results suggest that peptides with higher inhibitory potency are larger and have more negatively charged and hydrophilic residues No correlations were found between their IC50 values and other physicochemical and biochemical parameters, such as the number of positively charged residues, grand average of hydropathicity index and aliphatic index Identification of the potential binding site of FAS and inhibitory peptides from SBC Peptides EITPEKNPQLR and RKQEEDEDEEQQRE were selected for use in the ligand–enzyme docking simulations because they displayed higher FAS inhibitory potency To identify potential binding sites for these peptide inhibitors, the multidomain porcine FAS crystal structure (PBD ID code: 2VZ8) [25] was searched for cavities near the identified active site residues in each domain Because this structure lacked the ACP and TE domains, the human ACP structure (PBD ID code: 2CG5) and human TE domain structure (PBD ID code: 1XKT) lacking three short loop regions were also included in this search Modeling of these regions using the moe (Chemical Computing Group, Montreal, Canada) program allowed for cavity identification in the complete assembled structure, excluding openings extending into the structurally undefined interdomain regions Of the seven crystallographically defined domains, the 1484 Fig Identification of active-site cavities in fatty acid synthase (FAS) In this representation, the multidomain FAS is compiled in MOE from the swine FAS crystal structure (PDB ID code: 2VZ8) [25], the human ACP structure (PBD ID code: 2CG5) [41] and the human thioesterase (TE) domain structure (PBD ID code: 1XKT) [1] The protein backbone is represented as an orange line, active-site cavities are shown as blue spheres and catalytic residues are shown in space-filling format MAT, malonyl-CoA transacylase domain; KS, b-ketoacyl synthase domain; KR, b-ketoacyl reductase domain; DH, dehydratase domain; ER, b-enoyl reductase domain; ACP, acyl-carrier protein domain; TE, thioestherase domain TE domain had the largest cavity closest to the active ˚ site (3959 A3) and the b-enoyl reductase (ER) domain ˚ had the second largest cavity (3697 A3) (Fig 2) Docking of the EITPEKNPQLR inhibitory peptide in both sites predicted interaction energies in the ER domain higher than those in the TE domain (Table 3) Docking of the RKQEEDEDEEQQRE inhibitory peptide predicted near equivalent interaction energies in the ER and TE domains In both sites, the predicted energies of the protein–ligand complexes were much higher for the larger RKQEEDEDEEQQRE peptide than for the smaller EITPEKNPQLR peptide These results suggest that the ER domain is not flexible enough to accommodate relatively large inhibitors and that the TE domain is a better target for these types of peptide inhibitors To confirm the binding of these peptide inhibitors in the TE domain, a biochemical enzyme inhibition assay was performed using a recombinant human FAS TE The TE inhibitory activity displayed by peptides EITPEKNPQLR and RKQEEDEDEEQQRE was compared with C75 and Orlistat (Table 3) Soy peptides were more potent (10 lm) than C75 (58.7 lm), however, their potency was $ 10-fold lower (P < 0.05) than Orlistat (0.9 lm) Similar to C75, soy peptides blocked > 50% of the TE activity; however, this inhibition was lower (P < 0.05) than Orlistat (77.3%) at 100 lm Binding and interaction modes of FAS inhibitory peptides To evaluate in more detail the binding modes of these peptide inhibitors in the TE domain, they were FEBS Journal 277 (2010) 1481–1493 ª 2010 The Authors Journal compilation ª 2010 FEBS C Martinez-Villaluenga et al b-conglycinin peptides inhibit fatty acid synthase A B Subdomain B Loop II N His 2481 Asp 2338 Loop III Asp 2338 His 2481 Subdomain A Ser 2308 Ser 2308 Loop I C C Fig Predicted overall fold of the thioestherase (TE) domain with inhibitors bound The lowest-energy binding mode for EITPEKNPQLR (A), RKQEEDEDEEQQRE (B), C75 (C) and Orlistat (D) rendered in balland-stick format in the human TE domain model (backbone in tube format) is shown with catalytic triad residues Ser2308, His2481 and Asp 2338 in space-filling format Details regarding docking simulations are summarized in Table D His 2481 His 2481 Asp 2338 Asp 2338 Ser 2308 Ser 2308 Table Molecular docking within the fatty acid synthase (FAS) potential binding site and human FAS thioesterase (TE) inhibitory potency (IC50) of soybean b-conglycinin-derived peptides, C75 and Orlistat EITPEKNPQLR b-Enoyl reductase Predicted interaction energy (kcalỈmol)1) Predicted energy of protein–ligand complex (kcalỈmol)1) Thioesterase Predicted interaction energy (kcalỈmol)1) Predicted energy of protein–ligand complex (kcalỈmol)1) ˚ Distance to the catalytic triad (A) Ser2308 Asp2338 His2481 Inhibition (%) of human FAS TEb Human FAS TE inhibitory potency (IC50, lM)b RKQEEDEDEEQQRE C75a Orlistata )279.1 )1750.9 )438.0 )144.1 – – – – )353.0 )1180.3 )442.3 )955.5 )51.2 )980.44 )90.4 )1020.5 4.93 2.98 2.39 55.46 ± 1.45c 10.05 ± 1.60c 2.40 3.98 2.16 52.90 ± 4.34c 10.71 ± 4.36c 4.00 4.14 2.12 55.53 ± 2.43c 58.71 ± 6.74e 3.14 4.81 2.22 77.89 ± 0.81d 0.93 ± 0.13d a C75 and Orlistat were docked only in the active site of TE domain because they are known to target the TE domain [Cheng et al [31]] Compounds were tested at a concentration of 100 lM Values indicate the mean ± SD of at least two independent experiments c,d,e Different letters (c, d, e) in the same row indicate significant difference at P < 0.05 in one-way ANOVA analysis b docked individually within the predicted binding site using the DOCK function within moe and compared with the docking modes predicted for C75 and Orli- stat [14,26] The predicted lowest energy conformations of these peptides, C75 and Orlistat inhibitors, in the human FAS TE domain model are shown in FEBS Journal 277 (2010) 1481–1493 ª 2010 The Authors Journal compilation ª 2010 FEBS 1485 b-conglycinin peptides inhibit fatty acid synthase C Martinez-Villaluenga et al Fig Molecular surface representation of the thioestherase (TE) domain with inhibitory peptides EITPEKNPQLR (orange balland-stick format), RKQEEDEDEEQQRE (blue ball-and-stick format) and Orlistat (red ball-and-stick format) The potential surface is colored to reflect hydrogen bonding (pink), strong hydrophilic (green) and mild hydrophilic (blue) regions Fig 3A–D; interaction energies and the distances between inhibitor atoms and the catalytic triad are presented in Table In this docking mode, the EITPEKNPQLR peptide is predicted to be positioned ˚ at a distance of 4.93, 2.39 and 2.98 A from Ser2308, His2481 and Asp2338, respectively, in the TE domain (Fig 3A), with a low interaction energy ()353.0 kcalỈmol)1) (Table 3) By comparison, the larger RKQEEDEDEEQQRE peptide docked in the TE domain (Fig 3B) at distances of 2.40, 2.16 and ˚ 3.98 A from the Ser2308, His2481 and Asp2338, respectively, with the lowest interaction energy ()442.3 kcalỈmol)1), suggesting that this peptide is a better inhibitor than EITPEKNPQLR; this is in agreement with IC50 values listed in Table In their docking modes, C75 and Orlistat were positioned at greater distances from both the Asp2338 and Ser2308 residue in the TE domain than was the RKQEEDEDEEQQRE peptide and were predicted to interact more weakly ()51.2 and )90.4 kcalỈmol)1, respectively) with the TE domain than was the smaller SBC-derived peptide (Table 3) In addition, correlation analyses between the inhibitory potency of peptides EITPEKNPQLR, RKQEEDEDEEQQRE and C75 and their interaction energies with the TE domain showed a strong correlation (r = 0.99) Close-up views of the binding modes of SBC-derived peptides and Orlistat with the TE active site (Fig 4) suggest that the palmitic core of Orlistat is bound almost exclusively to a hydrophobic groove generated by subdomain B, and its peptidyl moiety is bound in the interface cavity, whereas the hexanoil tail digs into the short chain pocket where the catalytic triad exists These views also suggest that the larger EI1486 TPEKNPQLR and RKQEEDEDEEQQRE peptides bind throughout the long hydrophobic groove of the TE domain in a orientation similar to that of Orlistat with their amino acid side chains also extending into the interface cavity and short chain pocket The potential interaction modes of these peptides with the TE domain suggest that EITPEKNPQLR (Fig 5A) and RKQEEDEDEEQQRE (Fig 5B) bind mainly via hydrophilic interactions (hydrogen-bonding and electrostatic interactions) with active site residues By contrast, only the hydrophilic peptidyl group of Orlistat participates in hydrogen bonding with catalytic triad residues Tyr2307, His2481 and Arg2482 located in the interface cavity FAS inhibitory activity of synthetic peptides in 3T3-L1 adipocytes In a cell-based model, FAS inhibition was measured by monitoring the inhibition of lipid accumulation in 3T3-L1 adipocytes compared with the C75 positive control compound As shown in Fig 6, synthetic peptides displayed dose-dependent inhibition of lipid droplet accumulation in adipocytes The highest inhibition percentages for KNPQLR, EITPEKNPQLR and RKQEEDEDEEQQRE were observed after cell treatment at 100 lm (30.1, 29.6 and 34.2%, respectively) In these assays, KNPQLR and EITPEKNPQLR peptides showed similar (P > 0.05) inhibitory potency; however, significantly lower (P < 0.05) than C75 at 50 lm (38.8%) and 100 lm (46.3%) By contrast, the RKQEEDEDEEQQRE peptide showed an inhibitory activity similar to C75 at all concentrations tested with the exception of 50 lm, which displayed only 27.3% FEBS Journal 277 (2010) 1481–1493 ª 2010 The Authors Journal compilation ª 2010 FEBS C Martinez-Villaluenga et al b-conglycinin peptides inhibit fatty acid synthase A B C Fig Detailed 2D interactions between inhibitors and the thioestherase (TE) domain Calculated using the MOE program following the method of Clark & Labute [44], residues in the TE domain that contribute to the binding of EITPEKNPQLR (A), RKQEEDEDEEQQRE (B) and Orlistat (C) are shown with green circles indicating residues with no polar or charged side chains and light mauve circles indicating polar side chains that are either acidic (red ring) or basic (blue ring) Arrows indicate hydrogen bonds to side chain (green) and backbone (blue) residues inhibition (P > 0.05) Inhibition of lipid accumulation by these peptides correlated with FAS inhibition (r = 0.70) (Fig 6) even though the magnitudes of inhi- bition in these peptides in the cell-based model were lower than the magnitude of FAS inhibition measured in biochemical assays; this is probably because of the FEBS Journal 277 (2010) 1481–1493 ª 2010 The Authors Journal compilation ª 2010 FEBS 1487 b-conglycinin peptides inhibit fatty acid synthase C Martinez-Villaluenga et al 60 Inhibition (%) lipid accumulation KNPQLR EITPEKNPQLR 50 a ab RKQEEDEDEEQQRE C75 40 cde bc abc bcd 30 20 10 i ghi fgh hi def fghi fgh fg ef ghi 10 50 [compound] (µM) 100 Fig Inhibition of lipid accumulation in 3T3-L1 adipocytes by synthetic peptides 3T3-L1 adipocyte cells were treated with the synthetic KNPQLR, EITPEKNPQLR and RKQEEDEDEEQQRE peptides at concentrations ranging from to 100 lM on days 3, and 7, and lipid accumulation was measured on day 10 using the Oil Red O assay as outlined in Experimental procedures Each dataset corresponds to the average of three independent replicates with error bars indicating the standard deviation Different letters indicate significant differences at P < 0.05 in ANOVA analysis low permeability of cell to these longer peptides No effect on cell viability of 3T3-L1 adipocytes was observed with any of the treatments used in this study, indicating no cellular toxicity (data not shown) Discussion FAS is an important target for prevention and therapeutic interventions because multiple lines of evidence have shown high levels of FAS expression in cancer, obesity and metabolic disorders [27] The discovery and development of agents that block FAS activity highlight the potential for the prevention and treatment of those chronic diseases Our previous work demonstrated that SBC contains FAS inhibitory peptides that may be released by enzymatic hydrolysis with alcalase [23] This study has identified the FAS inhibitory peptides in the SBC hydrolysate using CIP, taking advantage of the specific affinity between FAS and its inhibitory peptides This CIP approach has identified for the first time three peptide fragments from the a and b subunits of SBC (KNPQLR, EITPEKNPQLR and RKQEEDEDEEQQRE) as potential inhibitors of FAS activity and their activities were confirmed using their custom synthesized peptides Our results have indicated that the inhibitory potency of these peptides (16.5–79.9 lm) is within the range found for purified SBC hydrolysates (IC50 = 30 lm) and soybean hydrolysates (50.4–175.1 lm) [23] In 1488 comparison with other natural inhibitors, the inhibitory potency of these peptides is within the range found for flavonoids from green tea (2.3–111.7 lm) [28,29] and tannins from Geum japonicum var chinense (0.2–41.4 lm) [22], which has been evaluated in preclinical studies [22,30] Molecular modeling has identified the TE domain as the potential binding site for the FAS inhibitory peptides from SBC Molecular docking has shown that soy peptides displayed a different inhibitory mechanism than C75 Soy peptides are selective inhibitors of the FAS TE domain, whereas C75 has been shown to interact at several sites in FAS [14] The predicted binding energy of C75 in the FAS b-ketoacyl synthase domain was )53.9 kcalỈmol)1, similar to that observed in the TE domain ()51.2 kcalỈmol)1) These results indicate that C75 is not a selective inhibitor for a particular FAS domain, in agreement with previous findings [14] We also confirmed that the synthetic peptides EITPEKNPQLR and RKQEEDEDEEQQRE inhibited 4-methylumbelliferone heptanoate hydrolysis by TE in in vitro experiments Therefore, these peptides are antagonists of TE under near physiologic conditions, meaning that they bind to the unoccupied enzyme and reduce substrate turnover The TE domain coordinates the terminal step of fatty acid synthesis by hydrolyzing palmitate from the 4¢-phosphopanteine arm of the ACP domain [1] Its active site is comprised of a hydrophobic groove with a distal pocket at the interface of subdomains A and B and a hydrophilic catalytic triad (Ser2308, His2481 and Asp2338) at the proximal end of the groove [17] Palmitate, the main biological product of FAS, binds in the hydrophobic groove, its hydrophilic carboxyl group interacting with the catalytic triad, and its hydrophobic, hydrocarbon chain extending away from the triad [31] From the binding modes that we have predicted, inhibitory peptides appear to block the catalytic activity of TE through hydrophilic interactions with enzyme residues located in the catalytic triad, the hydrophobic groove and the interface cavity The biochemical parameters of these peptides suggest that the numbers of negatively charged and hydrophylic residues are important predictors of their potency, in agreement with the fact that hydrophilic interactions are important to block the catalytic activity of the TE domain [31] The high number of charged and hydrophilic groups in these inhibitors provides for strong hydrogen bonding and electrostatic interactions with the catalytic residues of the TE domain Analysis of the TE domain in a variety of species indicates that catalytic triad residues are completely conserved from insects to mammals and all other FEBS Journal 277 (2010) 1481–1493 ª 2010 The Authors Journal compilation ª 2010 FEBS C Martinez-Villaluenga et al residues are conserved from birds to mammals, with the exception at Phe2370 which is changed to Ala2370 in chickens [1] This suggests that our current predictions on the binding mode of SBC-derived peptides in the human FAS TE domain can validly explain inhibition of catalytic activity in the chicken FAS Some evidence has shown that dietary soy protein may promote satiety and weight loss [32,33] and protect against certain types of cancer [34] The obesity-preventive effects of soybean protein have been associated with its ability to decrease lipid synthesis, adipogenesis and thermogenesis by regulating gene expression [32] Dietary intake of soy protein has also been reported to reduce tumor incidence in a rat model of chemically induced colon cancer by attenuating FAS expression [34] Our results provide additional insight into the preventive mechanisms of soy components in showing that SBC peptide fragments inhibit FAS activity in adipose cells in the same way as C75 Schmid et al [35] reported that FAS inhibition by C75 prevented adipogenesis in a cell-based model FAS inhibitory activity of SBC peptide fragments may potentially be found in cancer cells, liver or hypothalamus, as shown previously for C75 Inactivation of hypothalamic FAS by C75 is linked to satiety and dramatic weight loss [15] because accumulation of the substrate malonyl CoA through the inhibition of FAS appears to inhibit the expression of neuropeptide Y which promotes ingestion [9] Moreover, the FAS inhibitory activity of C75 induced apoptosis and prevented the growth of multiple tumor xenografts in vivo [36,37] Our findings clarify the mechanism linking FAS inhibition with the anti-obesity effects of soy protein-derived peptides In conclusion, the soy peptides EITPEKNPQLR and RKQEEDEDEEQQRE inhibited the TE domain and de novo fatty acid synthesis in adipocytes The binding mode of these peptides in the large palmitatebinding pocket is of particular interest and will guide future research These FAS inhibitory peptides can serve as lead compounds to design peptoid analogs (oligomers of N-subtituted glycine) with equivalent biological activity, enhanced systemic stability and bioavailability than standard peptides [38] The relevance of the identification of these SBC-derived peptides is noticeable because of the novelty of their biological activity and chemical nature Molecular docking has allowed us to predict binding modes for SBC-derived peptides (EITPEKNPQLR and RKQEEDEDEEQQRE) in the TE domain Based on our data, it is likely that the consumption of soy high in b-conglycinin represents a preventive alternative to improve health and wellness b-conglycinin peptides inhibit fatty acid synthase Experimental procedures Materials b-Conglycinin was purified from soybean defatted flour as described in Wang et al [39] FAS inhibitory peptides were produced from SBC hydrolysis with alcalase from Bacillus licheniformis, as detailed in Martinez-Villaluenga et al [24] The identified FAS inhibitory peptides (> 95% purity) were custom synthesized by GenScript (Piscataway, NJ, USA) FAS was isolated from chicken liver and purified (70% purity) as described by Tian et al [40] Human recombinant FAS TE (residues 2010–2509) was kindly provided by J.W Smith (Burnham Institute for Medical Research, CA, USA) 3T3-L1 (also designated ATCC CCL-92.1) preadipocytes from Swiss albino mouse and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from the American Type Culture Collection (Rockville, MD, USA) Calf bovine serum, fetal bovine serum and Dulbecco’s phosphate buffer saline were from Invitrogen (Rockville, MD, USA) Alcalase from B licheniformis (EC 3.4.21.62) and C75 were purchased from SigmaAldrich (St Louis, MO, USA) Protein A ⁄ G beads, nonspecific goat IgG and goat polyclonal IgG against a peptide mapping at the C-terminus of FAS were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) Unless otherwise stated, all chemical reagents were from Sigma-Aldrich FAS activity assay FAS activity was assayed by a spectrophotometric method using a Synergy Microplate Reader System equipped with temperature controller (Biotek Instruments, Winooksi, VA, USA) NADPH oxidation was followed at 37 °C by measuring the decrease in absorbance at 340 nm in a 96-well clear-bottomed polysterene plate (Corning, NY, USA) Reactions were performed in a final volume of 150 lL containing lm acetyl-CoA, 10 lm malonyl-CoA and 35 lm NADPH and 0.3 lm FAS in 0.1 m potassium phosphate buffer Initial rates were calculated for the slope of the progress curves during the first FAS inhibition studies Synthetic peptides and the C75 positive control compound were used for FAS inhibition studies with stock solutions of the synthetic peptides and C75 dissolved in deionized water and dimetylsulfoxide, respectively, and serial dilutions made in 0.1 m potassium phosphate buffer (pH 7.0) Inhibition studies were performed by measuring the residual FAS activity after enzyme preincubation with inhibitors for 20 at 37 °C Potency was determined by dose–response curves in which the range of concentrations was distributed in a logarithmic scale and the IC50 values were calculated FEBS Journal 277 (2010) 1481–1493 ª 2010 The Authors Journal compilation ª 2010 FEBS 1489 b-conglycinin peptides inhibit fatty acid synthase C Martinez-Villaluenga et al using nonlinear regression sigmoidal curve fit functions in GraphPad prism 4.00 (Graphpad Software Inc., San Diego, CA, USA) Inhibition of FAS TE enzymatic activity was performed using a fluorescence method described by Richardson & Smith [41] Peptides were added to yield a final concentration of 100 lm; in this assay the ability of the recombinant TE to cleave 4-methylumbelliferone heptanoate and hydrolyzed it to the fluorescent 4-methylumbelliferone was followed over time at 360 ⁄ 435 nm Co-immunoprecipitation To purify FAS inhibitory peptides a CIP approach was performed Briefly, 200 lL of b-conglycinin hydrolysate (2.5 mgỈmL)1 in 0.1 m potassium phosphate buffer, pH 7.0) were added with lL of goat IgG and 25 lL of protein A ⁄ G beads to preclear nonspecific peptides binding to IgG and ⁄ or agarose beads These samples were mixed on an end-over-end mixer for 60 at °C After preclearing, samples were centrifuged at 1000 g for at °C The supernatant (80 lL) was added to 120 lL of lm FAS in 0.1 m potassium phosphate buffer (pH 7.0) The negative control consisted of 80 lL of 0.1 m potassium phosphate buffer (pH 7.0) added to 120 lL FAS The blank consisted of 200 lL of 0.1 m potassium phosphate buffer (pH 7.0) These samples were incubated for 40 at 37 °C For CIP, each sample was incubated with 10 lL goat polyclonal antibody (FAS IgG) for 60 at °C and then 30 lL protein A ⁄ G beads were added and mixed on an end-over-end mixer overnight at °C After incubation with the antibody, samples were centrifuged at 1000 g for at °C and the pellet washed three times with radioimmunoprecipitation buffer The sediment was resuspended in HPLC-grade water and boiled for to release proteins from the beads Then, 20 lL acetonitrile containing 0.8 lL formic acid were added to extract the peptides and proteins, the beads were removed by centrifugation at 1000 g for at °C, and the final supernatant was stored at )20 °C before identification of FAS inhibitory peptides Western blot analysis To confirm the CIP of FAS, western blot analysis was carried out using goat polyclonal antibody (FAS IgG) Proteins released from the beads were resuspended in Laemmli loading buffer (BioRad, Hercules, CA, USA) containing 5% 2-mercaptoethanol Samples (20 lg soluble protein) were loaded onto 15% Tris ⁄ HCl ready gels and run through a mini-electrophoresis kit at 200 V constant for 40 Further, proteins were transferred to poly(vinylidene diflouride) membrane (BioRad) in blotting buffer (25 mm Tris, 192 mm glycine pH 8.3, 0.1% SDS) using western sandwich assembly for h at °C using 125 V 1490 After the transfer, membrane was blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for h, followed by an overnight incubation with goat polyclonal anti-(FAS IgG) (1 : 200) at °C Further, membrane was washed with TBST four times and was incubated with bovine anti-(goat IgG) horseradish peroxidase conjugates (1 : 1000) for h at room temperature The membrane was washed again in TBST for four times and signals were visualized using chemiluminescence reagent (GE Healthcare, Chalfont St Giles, UK) and a Kodak Image Station 440 CF (Eastman Kodak Co., New Haven, CT, USA) LC-MS ⁄ MS Samples were injected (10 lL) onto a dC18 Atlantis nanoAcquity column (75 · 150 mm, lm particle size; Waters, Milford, MA, USA) using 0.1% aqueous formic acid as solvent A and acetonitrile with 0.1% formic acid as solvent B A linear gradient from to 60% B was run for 60 and back to 1% B for 10 with the flow rate maintained at 0.25 mLỈmin)1 MS analysis was carried out in a Q-Tof API-US nanoAcquity LC (Waters) mass spectrometer equipped with an electron spray ion source The Q-Tof instrument was operated in positive ion mode Spectra were recorded over the m ⁄ z range 100–1500 Using MASCOT the m ⁄ z spectral data were processed and used for de novo peptide sequencing and database searching Peptide identification was carried out by searching against the NCBI or SWISS-PROT database [taxonomy = viridiplantae (green plants)] Only peptides identified with a confidence of at least 95% were considered to be correct calls (P < 0.05) Cell culture and treatments The 3T3-L1 preadipocytes were seeded at · 104 cellsỈwell)1 in 24-well plates and cultured in DMEM growth medium containing 1% sodium pyruvate, 1% penicillin ⁄ streptomycin and 10% calf bovine serum (days and 2) After reaching 100% confluence, the cells were stimulated with DMEM growth medium containing 1% sodium pyruvate, 1% penicillin ⁄ streptomycin, 10% fetal bovine serum, 0.5 mm isobutylmethylxanthine, lm dexamethasone and 1.7 lm insulin (days and 4) Cells were then maintained in fetal bovine serum ⁄ DMEM with 1.7 lm insulin for another days (days and 6), followed by culturing with fetal bovine serum ⁄ DMEM for an additional days (days 7–10), at which time > 90% of cells were mature adipocytes with fat droplets Cells were treated on days 3, and of the differentiation process with synthetic peptides dissolved in Dulbecco’s phosphate buffer saline at a concentration ranging from to 50 lm and incubated at 37 °C in a 5% CO2 atmosphere for 48 h FEBS Journal 277 (2010) 1481–1493 ª 2010 The Authors Journal compilation ª 2010 FEBS C Martinez-Villaluenga et al Viability assay The cell proliferation assay was conducted using CellTiter 96 Aqueous One Solution Proliferation assay kit (Promega Corp., Madison, WI, USA) using 3-(4,5-dimethylthiazol2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, and an electron coupling reagent, phenazine ethosulfate Briefly, · 103 preadipocytesỈwell)1 were seeded in a 96-well plate and the total volume was adjusted to 200 lL with growth medium Cells were treated on days 3, and of the differentiation process with different concentrations of synthetic peptides dissolved in Dulbecco’s phosphate buffer saline On day 10, the growth medium was replaced by 100 lL fresh growth medium and 20 lL 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium bromide ⁄ phenazine ethosulfate was added to each well The plate was incubated for h at 37 °C and the absorbance (A) was read at 515 nm in a microplate reader The percentage of viable cells was calculated with respect to cells treated with Dulbecco’s phosphate buffer saline using the following equation: Atreatment; 515 nm =Acontrol; 515 nm  100 ¼ % cell viability Lipid quantification in 3T3-L1 adipocytes by Oil Red O assay Adipocytes were washed twice with cold Dulbecco’s phosphate buffer saline and fixed with 10% formaldehyde for h Then, cells were washed with 60% isopropanol and air dried Oil Red O stock solution (0.2 g in 60% isopropanol) was filtered through a 0.22-lm membrane and added to lipid droplets for 30 After Oil Red O lipid staining, cells were washed with water four times and air dried Oil Red O dye was eluted by adding 100% isopropanol After 10 incubation at room temperature, the absorbance (A) at 510 nm was measured using a microplate reader Percent inhibition of lipid accumulation was calculated using the following equation:   Acontrol; 510 nm À Atreatment; 510 nm = Acontrol; 510 nm  100 ¼ % inhibition of lipid content b-conglycinin peptides inhibit fatty acid synthase the interdomain regions were excluded because they could not be accounted for with the single domain structures The volumes of each of the identified cavities were calculated using the SITE VOLUME SCRIPT function within moe The human TE domain structure available (PBD ID code: 1XKT) [1] lacked three loop regions: loop I (residues 2326– 2328 missing in chain A only) that connects a helix (a4) to b strand (b5) and forms a surface loop on the underside of the a ⁄ b domain; loop II (residues 2344–2360) that bridges subdomain A and subdomain B; and loop III (residues 2450–2460) that occurs near the catalytic triad linking b6 to b7 The missing loops were modeled using the HOMOLOGY function in moe 2008.10 and the aligned sequences of the TE domain of swine FAS (GenBank accession no NP_001093400) and human FAS (GenBank accession no AAB35516.1) Inhibitors were docked using the DOCK function of moe The initial 3D structures of the ligands were constructed using the BUILDER function in the moe program The initial positions of these compounds were set within the catalytic site and docking simulations were carried out by using the CHARMM27 force field [43] and the simulated annealing conformation search method within the DOCK function One hundred conformations were generated for each ligand tested while keeping protein side chains fixed, and these were sorted in ascending order according to their total energy Binding modes with the lowest total energies and extended conformation were chosen for second-round energy minimizations during which all protein side chains were allowed to move freely Protein–ligand interactions were established using the LIGAND INTERACTION function in moe which follows the method described by Clark & Lebute [44] Statistical analysis Data were expressed as means of at least two independent replicates Results were compared using one-way analysis of variance (ANOVA) using the GLM procedure of sas (SAS Institute, Cary, NC, USA) Group means were considered to be significantly different, as determined by the technique of protective least-significant differences (LSD), when ANOVA indicated an overall significant treatment effect (P < 0.05) Acknowledgements Molecular modeling To identify potential binding sites for the peptide inhibitors the multi domain porcine fatty acid synthase crystal structure (PBD ID code: 2VZ8) [25] was searched for cavities near the identified active site residues in each domain Because this structure lacked the ACP and TE domains, the human ACP structure (PBD ID code: 2CG5) [42] was used in the search Parts of identified cavities extending to This research was supported by the USDA Cooperative State Research, Education and Extension Service (CSREES), AG 2007-34505-15767 Future Foods IL; Illinois Soybean Association; the European Commission, Marie Curie IOF grant (PIOF-GA-2008-219860) for Career Development (to CM-V) Special acknowledgements to Drs J W Smith and R D Richardson FEBS Journal 277 (2010) 1481–1493 ª 2010 The Authors Journal compilation ª 2010 FEBS 1491 b-conglycinin peptides inhibit fatty acid synthase C Martinez-Villaluenga et al from the Burnham Institute for Medical Research, California for providing us with the human recombinant FAS TE References Chakravarty B, Gu ZW, Chirala SS, Wakil SJ & Quiocho FA (2004) Human fatty acid synthase: structure and substrate selectivity of the thioesterase domain Proc Natl Acad Sci USA 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(2007) Mechanism and substrate recognition of human holo ACP synthase Chem Biol 14, 1243–1253 43 MacKerell AD, Bashford D, Bellott M, Dunbrack RL, Evanseck JD, Field MJ, Fischer S, Gao J, Guo H, Ha S et al (1998) All-atom empirical potential for molecular modeling and dynamics studies of proteins J Phys Chem B 102, 3586–3616 44 Clark AM & Labute P (2007) 2D depiction of protein– ligand complexes J Chem Inf Model 47, 1933–1944 FEBS Journal 277 (2010) 1481–1493 ª 2010 The Authors Journal compilation ª 2010 FEBS 1493 ... et al b-conglycinin peptides inhibit fatty acid synthase A B C Fig Detailed 2D interactions between inhibitors and the thioestherase (TE) domain Calculated using the MOE program following the method... b-conglycinin peptides inhibit fatty acid synthase the interdomain regions were excluded because they could not be accounted for with the single domain structures The volumes of each of the identified... Table Molecular docking within the fatty acid synthase (FAS) potential binding site and human FAS thioesterase (TE) inhibitory potency (IC50) of soybean b-conglycinin- derived peptides, C75 and Orlistat