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Stuart Smith - One of the best experts on this subject based on the ideXlab platform.
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Conformational flexibility of metazoan Fatty Acid Synthase enables catalysis.
Nature structural & molecular biology, 2009Co-Authors: Edward J. Brignole, Stuart Smith, Francisco J. AsturiasAbstract:Fatty Acid Synthase is composed by several catalytic domains that work in sequence, with reaction intermediates being transferred between them. Single-particle EM analysis of different catalytic mutants of rat FAS imaged in the presence of substrates reveals the domains' movements during the reaction cycle.
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Effect of Modification of the Length and Flexibility of the Acyl Carrier Protein−Thioesterase Interdomain Linker on Functionality of the Animal Fatty Acid Synthase†
Biochemistry, 2005Co-Authors: Anil K. Joshi, Lei Zhang, Andrzej Witkowski, Harvey Alan Berman, Stuart SmithAbstract:A natural linker of approximately 20 residues connects the acyl carrier protein with the carboxy-terminal thioesterase domain of the animal Fatty Acid Synthase. This study examines the effects of changes in the length and amino Acid composition of this linker on catalytic activity, product composition, and segmental motion of the thioesterase domain. Deletion of 10 residues, almost half of the interdomain linker, had no effect on either mobility of the thioesterase domain, estimated from fluorescence polarization of a pyrenebutyl methylphosphono moiety bound covalently to the active site serine residue, or functionality of the Fatty Acid Synthase; further shortening of the linker limited mobility of the thioesterase domain and resulted in reduced Fatty Acid Synthase activity and an increase in product chain length from 16 to 18 and 20 carbon atoms. Surprisingly, however, even when the entire linker region was deleted, the Fatty Acid Synthase retained 28% activity. Lengthening of the linker, by insertion o...
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Cloning, expression, characterization, and interaction of two components of a human mitochondrial Fatty Acid Synthase. Malonyltransferase and acyl carrier protein.
Journal of Biological Chemistry, 2003Co-Authors: Lei Zhang, Anil K. Joshi, Stuart SmithAbstract:Abstract The possibility that human cells contain, in addition to the cytosolic type I Fatty Acid Synthase complex, a mitochondrial type II malonyl-CoA-dependent system for the biosynthesis of Fatty Acids has been examined by cloning, expressing, and characterizing two putative components. Candidate coding sequences for a malonyl-CoA:acyl carrier protein transacylase (malonyltransferase) and its acyl carrier protein substrate, identified by BLAST searches of the human sequence data base, were located on nuclear chromosomes 22 and 16, respectively. The encoded proteins localized exclusively in mitochondria only when the putative N-terminal mitochondrial targeting sequences were present as revealed by confocal microscopy of HeLa cells infected with appropriate green fluorescent protein fusion constructs. The mature, processed forms of the mitochondrial proteins were expressed in Sf9 cells and purified, the acyl carrier protein was converted to the holoform in vitro using purified human phosphopantetheinyltransferase, and the functional interaction of the two proteins was studied. Compared with the dual specificity malonyl/acetyltransferase component of the cytosolic type I Fatty Acid Synthase, the type II mitochondrial counterpart exhibits a relatively narrow substrate specificity for both the acyl donor and acyl carrier protein acceptor. Thus, it forms a covalent acyl-enzyme complex only when incubated with malonyl-CoA and transfers exclusively malonyl moieties to the mitochondrial holoacyl carrier protein. The type II acyl carrier protein from Bacillus subtilis, but not the acyl carrier protein derived from the human cytosolic type I Fatty Acid Synthase, can also function as an acceptor for the mitochondrial transferase. These data provide compelling evidence that human mitochondria contain a malonyl-CoA/acyl carrier protein-dependent Fatty Acid Synthase system, distinct from the type I cytosolic Fatty Acid Synthase, that resembles the type II system present in prokaryotes and plastids. The final products of this system, yet to be identified, may play an important role in mitochondrial function.
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Structural and functional organization of the animal Fatty Acid Synthase.
Progress in lipid research, 2003Co-Authors: Stuart Smith, Andrzej Witkowski, Anil K. JoshiAbstract:Abstract The entire pathway of palmitate synthesis from malonyl-CoA in mammals is catalyzed by a single, homodimeric, multifunctional protein, the Fatty Acid Synthase. Each subunit contains three N-terminal domains, the β-ketoacyl Synthase, malonyl/acetyl transferase and dehydrase separated by a structural core from four C-terminal domains, the enoyl reductase, β-ketoacyl reductase, acyl carrier protein and thiosterase. The kinetics and specificities of the substrate loading reaction catalyzed by the malonyl/acetyl transferase, the condensation reaction catalyzed by β-ketoacyl Synthase and chain-terminating reaction catalyzed by the thioesterase ensure that intermediates do not leak off the enzyme, saturated chains exclusively are elongated and palmitate is released as the major product. Only in the Fatty Acid Synthase dimer do the subunits adopt conformations that facilitate productive coupling of the individual reactions for Fatty Acid synthesis at the two acyl carrier protein centers. Introduction of a double tagging and dual affinity chromatographic procedure has permitted the engineering and isolation of heterodimeric Fatty Acid Synthases carrying different mutations on each subunit. Characterization of these heterodimers, by activity assays and chemical cross-linking, has been exploited to map the functional topology of the protein. The results reveal that the two acyl carrier protein domains engage in substrate loading and condensation reactions catalyzed by the malonyl/acetyl transferase and β-ketoacyl Synthase domains of either subunit. In contrast, the reactions involved in processing of the β-carbon atom, following each chain elongation step, together with the release of palmitate, are catalyzed by the cooperation of the acyl carrier protein with catalytic domains of the same subunit. These findings suggest a revised model for the Fatty Acid Synthase in which the two polypeptides are oriented such that head-to-tail contacts are formed both between and within subunits.
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Fatty Acid Synthase: in vitro complementation of inactive mutants.
Biochemistry, 1996Co-Authors: Andrzej Witkowski, And Anil K. Joshi, Stuart SmithAbstract:The animal Fatty Acid Synthase is a dimer of identical, multifunctional 272 kDa subunits oriented antiparallel such that two centers for Fatty Acid synthesis are formed at the subunit interface. In order to clarify the interdomain and intersubunit communications necessary for the operation of the two centers, we have explored the possibility of reassembling catalytically-active Fatty Acid Synthase heterodimers from pairs of inactive dimers carrying mutations in different functional domains. To this end, rat Fatty Acid Synthase mutants, defective in either the beta-ketoacyl Synthase, C161T or K326A (KS- FAS), or the acyl carrier protein, S2151A (ACP- FAS), domains, were engineered by site-directed mutagenesis, expressed in insect Sf9 cells using a baculovirus expression system, and purified. A novel procedure was devised to facilitate rapid production and isolation of a population of mixed mutant dimers that had undergone randomization of its constituent subunits. Homodimeric mutants (KS- FAS/KS- FAS and ACP- FAS/ACP- FAS) and KS- FAS heterodimers consisting of paired C161T and K326A mutant subunits were unable to synthesize Fatty Acids, confirming the essential nature of residues C161, K326, and S2151A. However, KS- FAS/ACP- FAS heterodimers regained partial activity. Formation of these heterodimers necessitated prior dissociation and reassociation of the homodimers, indicating that the rate of spontaneous exchange of subunits in the dimer is negligible. The formation of catalytically-active heterodimers from pairs of inactive, complementary homodimers affords a useful method for testing the validity of the current model for the multifunctional complex.
Steven D. Clarke - One of the best experts on this subject based on the ideXlab platform.
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Involvement of a Unique Carbohydrate-responsive Factor in the Glucose Regulation of Rat Liver Fatty-Acid Synthase Gene Transcription
The Journal of biological chemistry, 2001Co-Authors: Caterina Rufo, Margarita Teran-garcia, Manabu T. Nakamura, Seung Hoi Koo, Howard C. Towle, Steven D. ClarkeAbstract:Abstract Refeeding carbohydrate to fasted rats induces the transcription of genes encoding enzymes of Fatty Acid biosynthesis,e.g. Fatty-Acid Synthase (FAS). Part of this transcriptional induction is mediated by insulin. An insulin response element has been described for the Fatty-Acid Synthase gene region of −600 to +65, but the 2–3-fold increase in Fatty-Acid Synthase promoter activity attributable to this region is small compared with the 20–30-fold induction in Fatty-Acid Synthase gene transcription observed in fasted rats refed carbohydrate. We have previously reported that the Fatty-Acid Synthase gene region between −7382 and −6970 was essential for achieving high in vivorates of gene transcription. The studies of the current report demonstrate that the region of −7382 to −6970 of the Fatty-Acid Synthase gene contains a carbohydrate response element (CHO-REFAS) with a palindrome sequence (CATGTGn 5GGCGTG) that is nearly identical to the CHO-RE of the l-type pyruvate kinase and S14 genes. The glucose responsiveness imparted by CHO-REFAS was independent of insulin. Moreover, CHO-REFAS conferred glucose responsiveness to a heterologous promoter (i.e. l-type pyruvate kinase). Electrophoretic mobility shift assays demonstrated that CHO-REFAS readily bound a unique hepatic ChoRF and that CHO-REFAS competed with the CHO-RE of thel-type pyruvate kinase and S14 genes for ChoRF binding. In vivo footprinting revealed that fasting reduced and refeeding increased ChoRF binding to CHO-REFAS. Thus, carbohydrate responsiveness of rat liver Fatty-Acid Synthase appears to require both insulin and glucose signaling pathways. More importantly, a unique hepatic ChoRF has now been shown to recognize glucose responsive sequences that are common to three different genes: Fatty-Acid Synthase, l-type pyruvate kinase, and S14.
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Arachidonic Acid suppression of Fatty Acid Synthase gene expression in cultured rat hepatocytes.
Biochemical and biophysical research communications, 1991Co-Authors: Michael K. Armstrong, William L. Blake, Steven D. ClarkeAbstract:Abstract Rat hepatocytes were maintained in a serum-free, hormonally defined medium supplemented with 50–500 μM albumin-bound 20:1 (n-9) vs 20:4 (n-6). The induction of Fatty Acid Synthase mRNA by a mix of insulin/dexamethasone/T3 was inhibited in a dose dependent fashion by 20:4 (n-6). The abundance of β-actin mRNA was not suppressed by 20:4 (n-6). The expression of Fatty Acid Synthase was actually stimulated 2-fold by 20:1 (n-9). It would appear that the in vivo inhibition of Fatty Acid Synthase gene expression by dietary polyunsaturated Fatty Acids is a specific hepatocelluar event.
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Porcine Fatty Acid Synthase: Cloning of a Complementary DNA, Tissue Distribution of Its mRNA and Suppression of Expression by Somatotropin and Dietary Protein
The Journal of nutrition, 1991Co-Authors: Ana M. Mildner, Steven D. ClarkeAbstract:A cDNA for porcine Fatty Acid Synthase was isolated and used to examine the tissue distribution of Fatty Acid Synthase mRNA within the pig and to determine the impact of recombinant porcine somatotropin (rpSt) and the level of dietary protein on Fatty Acid Synthase mRNA abundance in pig liver and adipose tissue. A 1.5-kb cDNA representing the thioesterase domain of porcine Fatty Acid Synthase was isolated from a lambda gt 11 liver cDNA library. Northern analysis with total RNA extracted from adipose tissue, liver, heart, lung, kidney and intestine revealed a single major Fatty Acid Synthase mRNA species of 8-9 kb. The amount of Fatty Acid Synthase mRNA in hepatic tissue was 25% of the amount in adipose tissue, which suggests that the liver may be a significant site of Fatty Acid synthesis in the pig. Fatty Acid Synthase mRNA abundance was significantly reduced in the adipose tissue (P less than 0.01) and the liver (P less than 0.1) by chronic daily administration (60 micrograms/kg) of rpSt. In addition, increasing the amount of dietary protein decreased (P less than 0.1) the abundance of Fatty Acid Synthase mRNA in adipose tissue but had no effect on liver Fatty Acid Synthase expression. In contrast, the abundance of adipose Fatty Acid binding protein mRNA was unaffected by rpSt or dietary protein. These data indicate that the reduction in the level of Fatty Acid Synthase mRNA is a factor in the pSt-mediated suppression of Fatty Acid synthesis in porcine adipose tissue.
Ruth Lupu - One of the best experts on this subject based on the ideXlab platform.
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Fatty Acid Synthase (FASN) as a therapeutic target in breast cancer.
Expert opinion on therapeutic targets, 2017Co-Authors: Javier A. Menendez, Ruth LupuAbstract:ABSTRACTIntroduction: Ten years ago, we put forward the metabolo-oncogenic nature of Fatty Acid Synthase (FASN) in breast cancer. Since the conception of this hypothesis, which provided a model to ...
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Fatty Acid Synthase and the lipogenic phenotype in cancer pathogenesis
Nature Reviews Cancer, 2007Co-Authors: Javier A. Menendez, Ruth LupuAbstract:Fatty Acid Synthase (FASN) catalyses the synthesis of Fatty Acids, and this synthetic pathway is upregulated in many tumours. How might FASN and increased lipogenesis be involved in cancer, and is FASN a valid therapeutic target? There is a renewed interest in the ultimate role of Fatty Acid Synthase (FASN) — a key lipogenic enzyme catalysing the terminal steps in the de novo biogenesis of Fatty Acids — in cancer pathogenesis. Tumour-associated FASN, by conferring growth and survival advantages rather than functioning as an anabolic energy-storage pathway, appears to necessarily accompany the natural history of most human cancers. A recent identification of cross-talk between FASN and well-established cancer-controlling networks begins to delineate the oncogenic nature of FASN-driven lipogenesis. FASN, a nearly-universal druggable target in many human carcinomas and their precursor lesions, offers new therapeutic opportunities for metabolically treating and preventing cancer.
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Oncogenic properties of the endogenous Fatty Acid metabolism: molecular pathology of Fatty Acid Synthase in cancer cells.
Current opinion in clinical nutrition and metabolic care, 2006Co-Authors: Javier A. Menendez, Ruth LupuAbstract:Purpose of reviewThis review documents our rapidly changing perspectives on the function of Fatty Acid Synthase-catalyzed endogenous Fatty Acid biogenesis in cancer biology.Recent findingsUp-regulation of Fatty Acid Synthase gene expression and Fatty Acid Synthase biosynthetic activity are molecular
Wei Xi Tian - One of the best experts on this subject based on the ideXlab platform.
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inhibitory effects of allium vegetable extracts on Fatty Acid Synthase
Food Science and Technology Research, 2009Co-Authors: Xuebing Sun, Wei Xi TianAbstract:Allium vegetables have been shown to have beneficial effects against several diseases, including hyperlipidemia and cancer. This study was carried out with enzymatic method to investigate the inhibition on Fatty Acid Synthase by nine Allium vegetables to explore their health protective mechanisms. It was found the Fatty Acid Synthase inhibitors in most Allium vegetables extracts were concentrated in the lower polar solvents and the inhibition could be reversed by dithiothreitol. Ethyl acetate extract of red onion (Allium cepa L., IC50 = 0.45 μg/mL) was the most active of the samples tested. In garlic, diallyl trisulfide was suggested to be an effective component which may react with the essential sulphydryl groups in the enzyme. Additionally, the Chinese chive (Allium tuberosum L.) exhibited the most total inhibitory capacity, and its inhibitory components showed more polar than that of other Allium vegetables. It is suggested that different inhibitors of FAS exist in these vegetables.
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keemun black tea extract contains potent Fatty Acid Synthase inhibitors and reduces food intake and body weight of rats via oral administration
Journal of Enzyme Inhibition and Medicinal Chemistry, 2005Co-Authors: Ya Tao Du, Xiao Dong Wu, Xuan Wang, Wei Xi TianAbstract:The inhibitory effects of a black tea extract on Fatty Acid Synthase were measured through inhibition kinetics. The Keemun black tea extract showed more potent inhibitory activity on Fatty Acid Synthase than green tea extract. Additionally, the inhibitory ability of the black tea extract depended on the extracting solvent and the conditions used. Only 10–23% of the inhibitory activity from the black tea was extracted by the general method of boiling with water. The results suggested that the main Fatty Acid Synthase inhibitors in black tea might be theaflavins. Which were more potent than epigallocatechin gallate or C75. The reaction site on the Fatty Acid Synthase and the inhibition kinetics for the extract were different from those of epigallocatechin gallate or C75. In addition, Keemun black tea extract significantly reduced food intake, body weight and blood triglyceride of diet-induced obesity SD rats via oral administration.
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Effects of osmolytes on unfolding of chicken liver Fatty Acid Synthase.
Biochemistry. Biokhimiia, 2002Co-Authors: Yong-doo Park, Wei Xi Tian, Hao ZhouAbstract:Urea-induced aggregation of chicken liver Fatty Acid Synthase [acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating,oxoacyl- and enoyl-reducing and thioester-hydrolyzing), EC 2.3.1.85 ] was studied. The aggregation was facilitated at increased ionic strength. Methyl-β-cyclodextrin and some osmolytes, such as glycerol, sucrose, proline, glycine, and heparin, could effectively prevent the aggregation, implying an artificial chaperone role of those substances during Fatty Acid Synthase unfolding. The osmolytes also protected the enzyme from inactivation.
Hai-meng Zhou - One of the best experts on this subject based on the ideXlab platform.
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The leaf extract of Siberian Crabapple (Malus baccata (Linn.) Borkh) contains potential Fatty Acid Synthase inhibitors.
Journal of enzyme inhibition and medicinal chemistry, 2008Co-Authors: Xiang Wei, Ran Zhao, Ying-hui Sun, Jian-ping Cong, Fan-guo Meng, Hai-meng ZhouAbstract:The present work focused on the kinetics of the inhibitory effects of the leaf extract of Siberian Crabapple, named Shan jingzi in China, on chicken liver Fatty Acid Synthase. The results showed that this extract had much stronger inhibitory ability on Fatty Acid Synthase than that from green teas described in many previous reports. The inhibitory ability of this extract is closely related to the extracting solvent, and the time of extraction was also an important influencing factor. The inhibitory types of this extract on diffeerent substrates of chicken liver Fatty Acid Synthase, acetyl-CoA, malonyl-CoA and NADPH, were found to be noncompetitive, uncompetitive and mixed, respectively. The studies here shed a new light on the exploration for inhibitors of Fatty Acid Synthase.
