Fatty acid synthase inhibition in human breast cancer cells leads to malonyl-CoA-induced inhibition of fatty acid oxidation and cytotoxicity.

Inhibition of fatty acid synthase (FAS) induces apoptosis in human breast cancer cells in vitro and in vivo without toxicity to proliferating normal cells. We have previously shown that FAS inhibition causes a rapid increase in malonyl-CoA levels identifying malonyl-CoA as a potential trigger of apoptosis. In this study we further investigated the role of malonyl-CoA during FAS inhibition. We have found that: [i] inhibition of FAS with cerulenin causes carnitine palmitoyltransferase-1 (CPT-1) inhibition and fatty acid oxidation inhibition in MCF-7 human breast cancer cells likely mediated by elevation of malonyl-CoA; [ii] cerulenin cytotoxicity is due to the nonphysiological state of increased malonyl-CoA, decreased fatty acid oxidation, and decreased fatty acid synthesis; and [iii] the cytotoxic effect of cerulenin can be mimicked by simultaneous inhibition of CPT-1, with etomoxir, and fatty acid synthesis with TOFA, an acetyl-CoA carboxylase (ACC) inhibitor. This study identifies CPT-1 and ACC as two new potential targets for cancer chemotherapy.     

Regulating effect of β-ketoacyl synthase domain of fatty acid synthase on fatty acyl chain length in de novo fatty acid synthesis

Fatty acid synthase (FAS) is a multifunctional homodimeric protein, and is the key enzyme required for the anabolic conversion of dietary carbohydrates to fatty acids. FAS synthesizes long-chain fatty acids from three substrates: acetyl-CoA as a primer, malonyl-CoA as a 2 carbon donor, and NADPH for reduction. The entire reaction is composed of numerous sequential steps, each catalyzed by a specific functional domain of the enzyme. FAS comprises seven different functional domains, among which the β-ketoacyl synthase (KS) domain carries out the key condensation reaction to elongate the length of fatty acid chain. Acyl tail length controlled fatty acid synthesis in eukaryotes is a classic example of how a chain building multienzyme works. Different hypotheses have been put forward to explain how those sub-units of FAS are orchestrated to produce fatty acids with proper molecular weight. In the present study, molecular dynamic simulation based binding free energy calculation and access tunnels analysis showed that the C16 acyl tail fatty acid, the major product of FAS, fits to the active site on KS domain better than any other substrates. These simulations supported a new hypothesis about the mechanism of fatty acid production ratio: the geometric shape of active site on KS domain might play a determinate role.     

Drosophila melanogaster fatty acid synthetase: Characteristics and effect of protease inhibitors

Fatty acid synthetase (FAS) from Drosophila melanogaster was purified by DEAE-cellulose and Sepharose CL-6B chromatography. Inclusion of protease inhibitors in all steps dramatically increased the specific activity of the FAS preparation (to an average value of 4500 U/mg protein, the highest value reported for any animal FAS). The relative molecular weight of the native enzyme was determined by gel filtration and found to be 480,000. SDS gel electrophoresis gave a subunit relative molecular weight of 226,000, indicating that D. melanogaster FAS, like other animal FASs, is a dimer. Acetyl-CoA was the most efficient primer with propionyl-CoA also supporting FAS activity. Neither hexanoyl-CoA butyryl-CoA, isobutyryl-CoA nor isovaleryl-CoA served as efficient primers. D. melanogaster FAS showed an absolute requirement for malonyl-CoA and no activity was observed when methylmalonyl-CoA replaced malonyl-CoA. However, in the presence of both elongating substrates, D. melanogaster FAS synthesized methyl branched fatty acids. Methylmalonyl-CoA appears to behave as a competitive inhibitor in the presence of malonyl-CoA     

Effects of mitochondrial uncoupling on adipocyte intracellular Ca(2+) and lipid metabolism

Previous data from this laboratory demonstrate that increased intracellular Ca(2+) ([Ca(2+)]i) coordinately regulates human and murine adipocyte lipid metabolism by stimulating lipogenesis and inhibiting lipolysis. However, recent data demonstrate metabolic uncoupling increases [Ca(2+)]i but inhibits lipogenesis by suppressing fatty acid synthase (FAS) activity. Accordingly, we have evaluated the interaction between mitochondrial uncoupling, adipocyte [Ca(2+)]i, and adipocyte lipid metabolism. Pretreatment of 3T3-L1 cells with mitochondrial uncouplers (DNP or FCCP) amplified the [Ca(2+)]i response to depolarization with KCl by 2-4 fold (p <0.001), while this increase was prevented by [Ca(2+)]i channel antagonism with lanthanum. Mitochondrial uncouplers caused rapid (within 4hr) dose-dependent inhibition of FAS activity (p <0.001), while lanthanum caused a further additive inhibition. The suppression of FAS activity induced by uncoupling was reversed by addition of ATP. Mitochondrial uncouplers increased FAS expression significantly while [Ca(2+)]i antagonism with lanthanum decreased FAS expression (P <0.001). In contrast, mitochondrial uncouplers independently inhibited basal and isoproterenol-stimulated lipolysis (20-40%, p <0.001), while this inhibition was fully reversed by lanthanum. Thus, mitochondrial uncoupling exerted short-term regulatory effects on adipocyte [Ca(2+)]i and lipogenic and lipolytic systems, serving to suppress lipolysis via a Ca(2+) -dependent mechanism and FAS activity via a Ca(2+)-independent mechanism.     

Zinc induces caspase-dependent mitochondrial pathway of the programmed cell death in haemocytes of Drosophila melanogaster

Zinc is an essential trace element in cells. However, its high level in cytoplasm promotes activation of stress signaling pathways and may lead to cell death. In the present study we used Drosophila melanogaster blood cells (haemocytes), obtained from the third instar larvae, to study the effects of high concentrations of Zn(2+) on programmed cell death (PCD). We analyzed the activity of caspases, the level of caspase inhibitor protein DIAP1 and metallothioneins, as well as calcium concentrations and activity of mitochondria in haemocytes exposed to 0.35 and 1.7 mM concentrations of Zn. The obtained results showed that rapid increase of [Zn(2+)]( i ) in the cytoplasm up-regulates metallothionein MtnB but not MtnA gene expression in cells treated with Zn(2+) in both concentrations. Excess of Zn(2+) also induced activation of the initiator caspase Dronc, associated with the mitochondrial pathway of PCD, and the effector caspase DrICE. In turn, the activity of receptor-regulated Dredd caspase was not changed. The level of DIAP1 decreased significantly in haemocytes in the presence of high Zn(2+) concentration in comparison to untreated cells. Moreover, mitochondrial membrane potential was significantly decreased after exposure to Zn ions. These results indicate that high concentration of Zn(2+) in the cytoplasm of haemocytes induces PCD via a mitochondrial pathway and that caspases play a pivotal role in this process.     

Nature of the Fatty Acid Synthetase Systems in Parenchymal and Epidermal Cells of Allium porrum L. Leaves

Fatty acid synthesis was compared in cell-free extracts of epidermis and parenchyma of Allium porrum L. leaves. Parenchyma extracts had the major fatty acid synthetase (FAS) activity (70-90%) of the whole leaf; palmitic acid was also the major fatty acid synthesized when acetyl-coenzyme A (CoA) was the primer, but when acetyl-acyl carrier protein (ACP) was employed, C_18:0 and C_16:0 were synthesized in equal proportion. With the epidermal FAS system when either acetyl-CoA or acetyl-ACP was tested in the presence of labeled malonyl-CoA, palmitic acid was the only product synthesized. Specific activities of the FAS enzyme activities were determined in both tissue extracts.

The properties of malonyl-CoA:ACP transacylase were examined from the two different tissues. The molecular weights estimated by Sephadex G-200 chromatography were 38,000 for the epidermal enzyme and 45,000 for parenchymal enzyme. The optimal pH was for both enzymes 7.8 to 8.0 and the maximal velocity 0.4 to 0.5 micromoles per milligram protein per minute. These enzymes had different affinities for malonyl-CoA and ACP. For the malonyl-CoA:ACP transacylase of epidermis, the K_m values were 5.6 and 13.7 micromolar for malonyl-CoA and ACP, respectively, and 4.2 and 21.7 micromolar for the parenchymal enzyme. These results suggest that the FAS system in both tissues are nonassociated, that the malonyl-CoA:ACP transacylases are isozymes, and that both in epidermis and in parenchyma tissue two independent FAS system occur. Evidence would suggest that β-ketoacyl-ACP synthase II is present in the parenchymal cells but missing in the epidermal cell.

     

Evaluation of inhibition of fatty acid synthase by ursolic acid: Positive cooperation mechanism

The inhibitory effect of ursolic acid (UA) on fatty acid synthase (FAS, EC 2.3.1.85) was investigated. We found that UA potently inhibited the activity of FAS with a half-inhibitory concentration value (IC₅₀) of 6.0 μg/ml. The inhibition kinetic results showed that the inhibition of FAS by UA was competitive against acetyl-CoA and malonyl-CoA, but uncompetitive to NADPH. UA at low concentration slowly inactivated FAS, but FAS was fast inactivated by high concentration of UA in a positive cooperative manner. Moreover, NADPH significantly enhanced the inactivation of FAS by low concentration of UA, but NADPH slightly decreased the inactivation of FAS by high concentration of UA. Taken together, the results suggest that ursolic acid decreases the FAS activity through inactivation of acetyl/malonyl transferase. The combination of NADPH and KR domain promotes the inhibitory effect of UA on FAS.     

Inhibitory effects of tannic acid on fatty acid synthase and 3T3-L1 preadipocyte

Tannic acid is a hydrolyzable tannin that exists in many widespread edible plants with a variety of biological activities. In this study, we found that tannic acid potently inhibited the activity of fatty acid synthase (FAS) in a concentration-dependent manner with a half-inhibitory concentration value (IC₅₀) of 0.14 μM. The inhibition kinetic results showed that the inhibition of FAS by tannic acid was mixed competitive and noncompetitive manner with respect to acetyl-CoA and malonyl-CoA, but uncompetitive to NADPH. Tannic acid prevented the differentiation of 3T3-L1 pre-adipocytes, and thus repressed intracellular lipid accumulation. In the meantime, tannic acid decreased the expression of FAS and down-regulated the mRNA level of FAS and PPARγ during adipocyte differentiation. Further studies showed that the inhibitory effect of tannic acid did not relate to FAS non-specific sedimentation. Since FAS was believed to be a therapeutic target of obesity, these findings suggested that tannic acid was considered having potential in the prevention of obesity.     

Biosynthesis of branched-chain fatty acid in bacilli: FabD (malonyl-CoA:ACP transacylase) is not essential for in vitro biosynthesis of branched-chain fatty acids

It was found that the partially purified beta-ketoacyl-ACP synthase of Bacillus insolitus did not require the addition of FabD (malonyl-CoA:ACP transacylase, MAT) for the activity assay. This study therefore examined the necessity of FabD protein for in vitro branched-chain fatty acid (BCFA) biosynthesis by crude fatty acid synthetases (FAS) of Bacilli. To discover the involvement of FabD in the BCFA biosynthesis, the protein was removed from the crude FAS by immunoprecipitation. The His-tag fusion protein FabD of Bacillus subtilis was expressed in Escherichia coli and used for the preparation of antibody. The rabbit antibody raised against the expressed fusion protein specifically recognized the FabD in the crude FAS of B. subtilis. Evaluation of the efficacy of the immunoprecipitation showed that a trace of FabD protein was present in the antibody-treated crude FAS. However, this complete removal of FabD from the crude FAS did not abolish its BCFA biosynthesis, but only reduced the level to 50-60% of the control level for acyl-CoA primer and to 80% for alpha-keto-beta-methylvalerate primer. Furthermore, the FabD concentration did not necessarily correlate with the MAT specific activity in the enzyme fractions, suggesting the presence of another enzyme source of MAT activity. This study, therefore, suggests that FabD is not the sole enzyme source of MAT for in vitro BCFA biosynthesis, and implies the existence of a functional connection between fatty acid biosynthesis and another metabolic pathway.     

圆红冬孢酵母新颖脂肪酸合酶的重组表达、纯化与活性检测

脂肪酸合酶(Fatty acid synthase,FAS)催化乙酰辅酶A和丙二酸单酰辅酶A反应生成脂肪酸,是油脂合成代谢途径中最重要的酶之一。在高产油脂的圆红冬孢酵母Rhodosporidium toruloides中发现了一种新颖的FAS,它含两个亚基,与其他物种的FAS相比,具有独特的结构域组成,尤其是含两个酰基载体蛋白(ACP)结构域。由于ACP在脂肪酸合成反应中起辅因子作用,推测多个ACP有利于提高FAS的催化活性,为研究该FAS的生物化学和结构特征,构建了表达FAS两个亚基的载体,并转化大肠杆菌Escherichia coli BL21(DE3),含pET22b-FAS1和pET24-FAS2质粒的重组菌株ZWE06可同时高表达两个亚基,经硫酸铵沉淀、蔗糖密度梯度离心和阴离子交换层析纯化,得到的重组FAS比活力达到548 mU/mg。纯化的FAS复合物可用于后续酶动力学和蛋白结构研究,且表达与纯化方法的建立对研究其他ACP的功能具有参考价值。     

Purification and characterization of a 2-oxoglutarate-linked ATP-independent deacetoxycephalosporin C synthase of Streptomyces lactamdurans

The deacetoxycephalosporin C (DAOC) synthase (expandase) of Streptomyces lactamdurans was highly purified, as shown by SDS-PAGE and isoelectric focusing. The enzyme catalysed the oxidative ring expansion that converts penicillin N into DAOC. The enzyme was very unstable but could be partially stabilized in 25 mM-Tris/HCl, pH 9.0, in the presence of DTT (0.1 mM). The enzyme required 2-oxoglutarate, oxygen and Fe2+, but did not need ATP, ascorbic acid, Mg2+ or K+. The optimum temperature was between 25 and 30 degrees C. The DAOC synthase showed a high specificity for the penicillin substrate. Only penicillin N but not isopenicillin N, penicillin G or 6-aminopenicillanic acid served as substrates. 2-Oxoglutarate analogues were not used as substrates although 2-oxobutyrate and 3-oxoadipate inhibited the enzyme by 100% and 56% respectively. The enzyme was strongly inhibited by Cu2+, Co2+ and Zn2+. The apparent Km values for penicillin N, 2-oxoglutarate and Fe2+ were 52 microM, 3 microM and 71 microM respectively. The enzyme was a monomer with a molecular mass of 27,000 Da +/- 1,000.     

The role of hypothalamic malonyl-CoA in energy homeostasis

Energy balance is monitored by hypothalamic neurons that respond to peripheral hormonal and afferent neural signals that sense energy status. Recent physiologic, pharmacologic, and genetic evidence has implicated malonyl-CoA, an intermediate in fatty acid synthesis, as a regulatory component of this energy-sensing system. The level of malonyl-CoA in the hypothalamus is dynamically regulated by fasting and feeding, which alter subsequent feeding behavior. Fatty acid synthase (FAS) inhibitors, administered systemically or intracerebroventricularly to lean or obese mice, increase hypothalamic malonyl-CoA leading to the suppression of food intake. Conversely, lowering malonyl-CoA with an acetyl-CoA carboxylase (ACC) inhibitor or by the ectopic expression of malonyl-CoA decarboxylase in the hypothalamus increases food intake and reverses inhibition by FAS inhibitors. Physiologically, the level of hypothalamic malonyl-CoA appears to be determined through phosphorylation/dephosphorylation of ACC by AMP kinase in response to changes in the AMP/ATP ratio, an indicator of energy status. Recent evidence suggests that the brain-specific carnitine:palmitoyl-CoA transferase-1 (CPT1c) may be a regulated target of malonyl-CoA that relays the "malonyl-CoA signal" in hypothalamic neurons that express the orexigenic and anorexigenic neuropeptides that regulate food intake and peripheral energy expenditure. Together these findings support a role for malonyl-CoA as an intermediary in the control of energy homeostasis.     

Tolerance and specificity of recombinant 6-methylsalicyclic acid synthase

BACKGROUND: 6-Methylsalicylic acid synthase (MSAS), a fungal polyketide synthase from Penicillium patulum, is perhaps the simplest polyketide synthase that embodies several hallmarks of this family of multifunctional enzymes--a large multidomain protein, a high degree of specificity toward acetyl-CoA and malonyl-CoA substrates, chain length control, and regiospecific ketoreduction. MSAS has recently been functionally expressed in Escherichia coli and Saccharomyces cerevisiae, leading to the engineered biosynthesis of 6-methylsalicylic acid in these hosts. These developments have set the stage for detailed mechanistic studies of this model system. RESULTS: A three--step purification procedure was developed to obtain >95% pure MSAS from extracts of E. coli. As reported earlier for the enzyme isolated from P. patulum, the recombinant enzyme produced 6-methylsalicylic acid (a reduced tetraketide) in the presence of acetyl-CoA, malonyl-CoA, and NADPH, but triacetic acid lactone (an unreduced triketide) in the absence of NADPH. Consistent with this observation, point mutations in the highly conserved nucleotide-binding motif of the ketoreductase domain also led to production of triacetic acid lactone in vivo. The enzyme showed some tolerance toward nonnatural primer units including propionyl- and butyryl-CoA, but was incapable of incorporating extender units from (R, S)-methylmalonyl-CoA. Interestingly, MSAS readily accepted the N-acetylcysteamine (NAC) analog of malonyl-CoA as a substrate. CONCLUSIONS: NAC thioesters are simple, cost-effective analogs of CoA thioester substrates, and therefore provide a facile strategy for probing the molecular recognition features of polyketide synthases using unnatural building blocks. The ability to produce 4-hydroxy-6-methyl-2-pyrone in both E. coli and yeast illustrates the feasibility of metabolic engineering of these hosts to produce unnatural polyketides. Finally, the abundant source of recombinant MSAS described here provides an opportunity to study this fascinating model system using a combination of structural, mechanistic, and mutagenesis approaches.     

Improved Phos-tag SDS-PAGE under neutral pH conditions for advanced protein phosphorylation profiling

We describe an improved Phos-tag SDS-PAGE (Zn(2+)-Phos-tag SDS-PAGE) using a dizinc(II) complex of Phos-tag acrylamide in conjunction with a Bis-tris-buffered neutral-pH gel system to detect shifts in the mobility of phosphoproteins. An existing technique (Mn(2+)-Phos-tag SDS-PAGE) using a polyacrylamide-bound Mn(2+)-Phos-tag and a conventional Laemmli's buffer system under alkaline pH conditions has limitations for separating certain phosphoproteins. The major improvements were demonstrated by visualizing novel up-shifted bands of commercially available pepsin, recombinant Tau treated in vitro with tyrosine kinases, and endogeneous β-catenin in whole-cell lysates. Additionally, the Zn(2+)-Phos-tag SDS-PAGE gels showed better long-term stability than the Mn(2+)-Phos-tag SDS-PAGE gels. We can therefore provide a simple, convenient, and more reliable homemade gel system for phosphate-affinity SDS-PAGE.     

Determination of malonyl-coenzyme A in rat heart,kidney, and liver: A comparison between acetyl-coenzyme A and butyryl-coenzyme A as fatty acid synthase primers in the assay procedure

The malonyl-CoA assay was nonlinear at low malonyl-CoA concentrations when labeled acetyl-CoA was used as fatty acid synthase primer. Linearity was obtained with low concentrations of both fatty acid synthase and labeled acetyl-CoA, but then the assay was disturbed by the diluting effect of endogenous acetyl-CoA. The problems of nonlinearity and dilution of radioactivity by endogenous compounds were absent when labeled butyryl-CoA was used as primer. The levels of malonyl-CoA in rat heart, kidney, and liver were determined. The use of butyryl-CoA gave higher values of malonyl-CoA.     

Suppression of fatty acid synthase, differentiation and lipid accumulation in adipocytes by curcumin

Curcumin is a well-known component of the cook seasoning and traditional herb turmeric (Curcuma longa), which has been reported to prevent obesity. However, the mechanism still remains to be determined. In this study, curcumin is found to be an effective inhibitor of fatty acid synthase (FAS), and its effects on adipocytes are further evaluated. Curcumin shows both fast-binding and slow-binding inhibitions to FAS. Curcumin inhibits FAS with an IC?? value of 26.8 μM, noncompetitively with respect to NADPH, and partially competitively against both substrates acetyl-CoA and malonyl-CoA. This suggests that the malonyl/acetyl transferase domain of FAS possibly is the main target of curcumin. The time-dependent inactivation shows that curcumin inactivates FAS with two-step irreversible inhibition, a specific reversible binding followed by an irreversible modification by curcumin. Like other classic FAS inhibitors, curcumin prevents the differentiation of 3T3-L1 cells, and thus represses lipid accumulation. In the meantime, curcumin decreases the expression of FAS, down-regulates the mRNA level of PPARγ and CD36 during adipocyte differentiation. Curcumin is reported here as a novel FAS inhibitor, and it suppresses adipocyte differentiation and lipid accumulation, which is associated with its inhibition of FAS. Hence, curcumin is considered to be having potential application in the prevention of obesity.     

Synthesis of methyl-branched fatty acids from methylmalonyl-CoA by fatty acid synthase from both the liver and the harderian gland of the guinea pig

Partially purified fatty acid synthase preparations from both the liver and the harderian gland of guinea pig showed the same relative rates of utilization of methylmalonyl-CoA when compared to malonyl-CoA. Radio gas-liquid chromatographic analysis of the products generated from [methyl-14C]methylmalonyl-CoA and from [2-14C]malonyl-CoA in the presence of unlabeled methylmalonyl-CoA showed that the enzyme from both tissues generated identical mixtures of branched fatty acids. Therefore, it is concluded that the production of methyl-branched acids only by the harderian gland is not due to any unique specificity of the fatty acid synthase of this gland, in contrast to the conclusion reached from results obtained from mass spectrometry of the products generated by crude extracts (Y. Seyama, H. Otsuka, A. Kawaguchi, and T. Yamakawa J. Biochem. 90, 789-797, 1981).     

Normal flux through ATP-citrate lyase or fatty acid synthase is not required for glucose-stimulated insulin secretion

It has been proposed that de novo synthesis of long-chain acyl-CoA (LC-CoA) is a signal for glucose-stimulated insulin secretion (GSIS). Key enzymes involved in synthesis of fatty acids from glucose include ATP-citrate lyase (CL) and fatty acid synthase (FAS). An inhibitor of CL, hydroxycitrate (HC), has been reported to inhibit insulin secretion in some laboratories but not in others. Here we show that high concentrations of NaCl created during preparation of HC by standard methods explain the inhibition of GSIS, and that removal of the excess NaCl prevents the effect. To further investigate the role of CL, two small interfering RNA adenoviruses (Ad-siCL2 and Ad-siCL3) were generated. Ad-siCL3 reduced CL mRNA levels by 92 +/- 6% and CL protein levels by 75 +/- 4% but did not affect GSIS in 832/13 cells compared with cells treated with a control adenovirus (Ad-siControl). Similar results were obtained with Ad-siCL2. Ad-siCL3-treated cells also exhibited a 52 +/- 7% reduction in cytosolic oxaloacetate, an 83 +/- 4% reduction in malonyl-CoA levels, and inhibition of [U-(14)C]glucose incorporation into lipid by 43 +/- 4%, all expected metabolic out-comes of CL suppression. Similarly, treatment of 832/13 cells with a recombinant adenovirus specific to FAS (Ad-siFAS) reduced FAS mRNA levels by 81 +/- 2% in 832/13 cells, resulting in a 59 +/- 4% decrease in [U-(14)C]glucose incorporation into lipid, without affecting GSIS. Finally, treatment of primary rat islets with Ad-siCL3 or Ad-siFAS reduced CL and FAS mRNA levels by 65 +/- 4% and 52 +/- 3%, respectively, but had no effect on GSIS relative to Ad-siControl-treated islets. These findings demonstrate that a normal rate of flux of glucose carbons through CL and FAS is not required for GSIS in insulinoma cell lines or rat islets.