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1.
J Am Chem Soc ; 143(23): 8533-8537, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-33978402

RESUMO

The de novo formation of lipid membranes from minimal reactive precursors is a major goal in synthetic cell research. In nature, the synthesis of membrane phospholipids is orchestrated by numerous enzymes, including fatty acid synthases and membrane-bound acyltransferases. However, these enzymatic pathways are difficult to fully reproduce in vitro. As such, the reconstitution of phospholipid membrane synthesis from simple metabolic building blocks remains a challenge. Here, we describe a chemoenzymatic strategy for lipid membrane generation that utilizes a soluble bacterial fatty acid synthase (cgFAS I) to synthesize palmitoyl-CoA in situ from acetyl-CoA and malonyl-CoA. The fatty acid derivative spontaneously reacts with a cysteine-modified lysophospholipid by native chemical ligation (NCL), affording a noncanonical amidophospholipid that self-assembles into micron-sized membrane-bound vesicles. To our knowledge, this is the first example of reconstituting phospholipid membrane formation directly from acetyl-CoA and malonyl-CoA precursors. Our results demonstrate that combining the specificity and efficiency of a type I fatty acid synthase with a highly selective bioconjugation reaction provides a biomimetic route for the de novo formation of membrane-bound vesicles.


Assuntos
Ácido Graxo Sintase Tipo I/metabolismo , Fosfolipídeos/biossíntese , Ácido Graxo Sintase Tipo I/química , Estrutura Molecular , Fosfolipídeos/química
2.
Protein Sci ; 29(2): 589-605, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31811668

RESUMO

De novo fatty acid biosynthesis in humans is accomplished by a multidomain protein, the Type I fatty acid synthase (FAS). Although ubiquitously expressed in all tissues, fatty acid synthesis is not essential in normal healthy cells due to sufficient supply with fatty acids by the diet. However, FAS is overexpressed in cancer cells and correlates with tumor malignancy, which makes FAS an attractive selective therapeutic target in tumorigenesis. Herein, we present a crystal structure of the condensing part of murine FAS, highly homologous to human FAS, with octanoyl moieties covalently bound to the transferase (MAT-malonyl-/acetyltransferase) and the condensation (KS-ß-ketoacyl synthase) domain. The MAT domain binds the octanoyl moiety in a novel (unique) conformation, which reflects the pronounced conformational dynamics of the substrate-binding site responsible for the MAT substrate promiscuity. In contrast, the KS binding pocket just subtly adapts to the octanoyl moiety upon substrate binding. Besides the rigid domain structure, we found a positive cooperative effect in the substrate binding of the KS domain by a comprehensive enzyme kinetic study. These structural and mechanistic findings contribute significantly to our understanding of the mode of action of FAS and may guide future rational inhibitor designs.


Assuntos
Caprilatos/química , Ácido Graxo Sintase Tipo I/química , Animais , Sítios de Ligação , Caprilatos/metabolismo , Ácido Graxo Sintase Tipo I/metabolismo , Ácidos Graxos/biossíntese , Ácidos Graxos/química , Cinética , Camundongos , Modelos Moleculares , Estrutura Molecular
3.
Sci Rep ; 9(1): 12987, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31506493

RESUMO

During fatty acid biosynthesis, acyl carrier proteins (ACPs) from type I fungal fatty acid synthase (FAS) shuttle substrates and intermediates within a reaction chamber that hosts multiple spatially-fixed catalytic centers. A major challenge in understanding the mechanism of ACP-mediated substrate shuttling is experimental observation of its transient interaction landscape within the reaction chamber. Here, we have shown that ACP spatial distribution is sensitive to the presence of substrates in a catalytically inhibited state, which enables high-resolution investigation of the ACP-dependent conformational transitions within the enoyl reductase (ER) reaction site. In two fungal FASs with distinct ACP localization, the shuttling domain is targeted to the ketoacyl-synthase (KS) domain and away from other catalytic centers, such as acetyl-transferase (AT) and ER domains by steric blockage of the KS active site followed by addition of substrates. These studies strongly suggest that acylation of phosphopantetheine arm of ACP may be an integral part of the substrate shuttling mechanism in type I fungal FAS.


Assuntos
Candida albicans/enzimologia , Microscopia Crioeletrônica/métodos , Ácido Graxo Sintase Tipo I/química , Ácido Graxo Sintase Tipo I/metabolismo , Conformação Proteica , Saccharomyces cerevisiae/enzimologia , Acilação , Sítios de Ligação , Domínio Catalítico , Modelos Moleculares , Transporte Proteico
4.
J Cell Biochem ; 120(10): 16643-16657, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31095793

RESUMO

For the past few decades, structure-based drug discovery (SBDD) has become an inevitable technique in the drug development process for screening hit compounds against therapeutic targets. Here, we have successfully used the SBDD approach viz. virtual high-throughput screening to identify potential inhibitors against the Ketoacyl synthase (KS) domain of Fatty acid synthase (FASN). Overexpression of FASN, and subsequent enhancement of de novo lipogenesis is a key survival strategy of cancer cells. Hence, targeting lipid metabolism using FASN inhibitors has been considered as a promising method to induce metabolic stress, thereby posing a survival disadvantage to cancer cells. In the present study, we have successfully identified eight FASN inhibitors from Asinex Elite database by implementing in silico tools. Five of the hit compounds share a common ring structure, which enables characteristic binding interactions with FASN-KS. Among them, in vitro validation showed that SFA 22637550 possesses significant FASN inhibitory activity and antiproliferative effect in human cancer cells of various origins. The maximum sensitivity was exhibited towards HepG2 hepatocellular carcinoma cells (IC50 = 28 µM). The mode of cell death was found to be apoptosis with a significant increase in SubG0 population without affecting any other phases of the cell cycle. The current study puts forward an excellent core structure for the development of potent FASN inhibitors for successfully targeting cancer cell metabolism, thereby causing selective cell death.


Assuntos
Antineoplásicos , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Inibidores Enzimáticos , Ácido Graxo Sintase Tipo I , Proteínas de Neoplasias , Piridinas , Antineoplásicos/química , Antineoplásicos/farmacologia , Simulação por Computador , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Ácido Graxo Sintase Tipo I/química , Ácido Graxo Sintase Tipo I/metabolismo , Células Hep G2 , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Piridinas/química , Piridinas/farmacologia
5.
Chembiochem ; 20(18): 2298-2321, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-30908841

RESUMO

De novo biosynthesis of fatty acids is an iterative process requiring strict regulation of the lengths of the produced fatty acids. In this review, we focus on the factors determining chain lengths in fatty acid biosynthesis. In a nutshell, the process of chain-length regulation can be understood as the output of a chain-elongating C-C bond forming reaction competing with a terminating fatty acid release function. At the end of each cycle in the iterative process, the synthesizing enzymes need to "decide" whether the growing chain is to be elongated through another cycle or released as the "mature" fatty acid. Recent research has shed light on the factors determining fatty acid chain length and has also achieved control over chain length for the production of the technologically interesting short-chain (C4 -C8 ) and medium-chain (C10 -C14 ) fatty acids.


Assuntos
Ácido Graxo Sintase Tipo II/química , Ácido Graxo Sintase Tipo I/química , Ácidos Graxos/biossíntese , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/genética , Animais , Bactérias/enzimologia , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo II/genética , Ácidos Graxos/química , Humanos , Estrutura Molecular , Plantas/enzimologia , Domínios Proteicos , Engenharia de Proteínas , Saccharomyces cerevisiae/enzimologia
6.
Protein Sci ; 28(2): 414-428, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30394635

RESUMO

Modularity is a fundamental property of megasynthases such as polyketide synthases (PKSs). In this study, we exploit the close resemblance between PKSs and animal fatty acid synthase (FAS) to re-engineer animal FAS to probe the modularity of the FAS/PKS family. Guided by sequence and structural information, we truncate and dissect animal FAS into its components, and reassemble them to generate new PKS-like modules as well as bimodular constructs. The novel re-engineered modules resemble all four common types of PKSs and demonstrate that this approach can be a powerful tool to deliver products with higher catalytic efficiency. Our data exemplify the inherent plasticity and robustness of the overall FAS/PKS fold, and open new avenues to explore FAS-based biosynthetic pathways for custom compound design.


Assuntos
Ácido Graxo Sintase Tipo I/química , Policetídeo Sintases/química , Engenharia de Proteínas , Dobramento de Proteína , Ácido Graxo Sintase Tipo I/genética , Humanos , Policetídeo Sintases/genética
7.
Sci Rep ; 8(1): 14864, 2018 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-30291265

RESUMO

The access to information on the dynamic behaviour of large proteins is usually hindered as spectroscopic methods require the site-specific attachment of biophysical probes. A powerful emerging tool to tackle this issue is amber codon suppression. Till date, its application on large and complex multidomain proteins of MDa size has not been reported. Herein, we systematically investigate the feasibility to introduce different non-canonical amino acids into a 540 kDa homodimeric fatty acid synthase type I by genetic code expansion with subsequent fluorescent labelling. Our approach relies on a microplate-based reporter assay of low complexity using a GFP fusion protein to quickly screen for sufficient suppression conditions. Once identified, these findings were successfully utilized to upscale both the expression scale and the protein size to full-length constructs. These fluorescently labelled samples of fatty acid synthase were subjected to initial biophysical experiments, including HPLC analysis, activity assays and fluorescence spectroscopy. Successful introduction of such probes into a molecular machine such as fatty acid synthases may pave the way to understand the conformational variability, which is a primary intrinsic property required for efficient interplay of all catalytic functionalities, and to engineer them.


Assuntos
Aminoácidos/genética , Códon de Terminação/genética , Ácido Graxo Sintase Tipo I/genética , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/genética , Sequência de Aminoácidos , Aminoácidos/química , Animais , Códon de Terminação/química , Ácido Graxo Sintase Tipo I/química , Código Genético , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Modelos Moleculares , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
9.
Nat Prod Rep ; 35(10): 1046-1069, 2018 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-30137093

RESUMO

Covering: up to mid of 2018 Type I fatty acid synthases (FASs) are giant multienzymes catalyzing all steps of the biosynthesis of fatty acids from acetyl- and malonyl-CoA by iterative precursor extension. Two strikingly different architectures of FAS evolved in yeast (as well as in other fungi and some bacteria) and metazoans. Yeast-type FAS (yFAS) assembles into a barrel-shaped structure of more than 2 MDa molecular weight. Catalytic domains of yFAS are embedded in an extensive scaffolding matrix and arranged around two enclosed reaction chambers. Metazoan FAS (mFAS) is a 540 kDa X-shaped dimer, with lateral reaction clefts, minimal scaffolding and pronounced conformational variability. All naturally occurring yFAS are strictly specialized for the production of saturated fatty acids. The yFAS architecture is not used for the biosynthesis of any other secondary metabolite. On the contrary, mFAS is related at the domain organization level to major classes of polyketide synthases (PKSs). PKSs produce a variety of complex and potent secondary metabolites; they either act iteratively (iPKS), or are linked via directed substrate transfer into modular assembly lines (modPKSs). Here, we review the architectures of yFAS, mFAS, and iPKSs. We rationalize the evolution of the yFAS assembly, and provide examples for re-engineering of yFAS. Recent studies have provided novel insights into the organization of iPKS. A hybrid crystallographic model of a mycocerosic acid synthase-like Pks5 yielded a comprehensive visualization of the organization and dynamics of fully-reducing iPKS. Deconstruction experiments, structural and functional studies of specialized enzymatic domains, such as the product template (PT) and the starter-unit acyltransferase (SAT) domain have revealed functional principles of non-reducing iterative PKS (NR-PKSs). Most recently, a six-domain loading region of an NR-PKS has been visualized at high-resolution together with cryo-EM studies of a trapped loading intermediate. Altogether, these data reveal the related, yet divergent architectures of mFAS, iPKS and also modPKSs. The new insights highlight extensive dynamics, and conformational coupling as key features of mFAS and iPKS and are an important step towards collection of a comprehensive series of snapshots of PKS action.


Assuntos
Ácido Graxo Sintase Tipo I/química , Policetídeo Sintases/química , Aciltransferases/química , Aciltransferases/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Evolução Molecular , Ácido Graxo Sintase Tipo I/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Filogenia , Policetídeo Sintases/metabolismo , Conformação Proteica , Metabolismo Secundário , Leveduras/enzimologia
10.
Chem Biol Drug Des ; 92(1): 1366-1372, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29635861

RESUMO

An Asinex Gold Platinium chemical library subset of 12 055 compounds was screened employing docking simulations in the active site of the human FAS KS domain. Among them, 13 compounds were further evaluated for their ability to inhibit fatty acid biosynthesis. Four compounds were found to be active in particular ASN05064661 and ASN05374526 with IC50 values of 6.6 and 10.5 µm, respectively. A binding mode study was further conducted with these two compounds structurally related to benzene sulfonamide and aromatic polyamide. This study showed that they fit tightly with the active site with several interactions, notably with the key residues Cys161, His293, and His331.


Assuntos
Ácido Graxo Sintase Tipo I/metabolismo , Ácidos Graxos/biossíntese , Bibliotecas de Moléculas Pequenas/química , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ácido Graxo Sintase Tipo I/química , Humanos , Lipogênese/efeitos dos fármacos , Camundongos , Simulação de Acoplamento Molecular , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia
11.
J Phys Chem B ; 122(1): 77-85, 2018 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-29210581

RESUMO

Human fatty acid synthase (hFAS) is a megasynthase whose main function is de novo biosynthesis of saturated fatty acids. Interest has been drawn to this enzyme beyond its physiological role due to the association between high levels of hFAS and clinical conditions such as obesity, diabetes, and cancer. Thus, it has become an undeniably attractive pharmacological target. Until now, no crystal structure of the complete hFAS is available, hindering attempts to fully understand this protein. Using homology modeling, we built a model of the entire megasynthase, encompassing all of its domains, including the acyl carrier protein (ACP) and thioesterase (TE) mobile domains absent in the crystal structure of mammalian fatty acid synthase (FAS). On a second stage, we used data-driven protein-protein docking between the substrate shuttling domain ACP and every catalytic domain in the protein. We also propose sets of amino acids at the interface of each domain that we believe are important to favor the interaction between ACP and each domain of hFAS. After inspection, we validated each complex between ACP and MAT/KS/KR/DH/ER domains through classical molecular dynamics simulations and RMSd analysis. Additionally, we mapped the interactions between the residues at the active site of each catalytic domain and its intermediaries. In every docking, we ensured that the distance between catalytic residues and the intermediaries was maintained. Until now, there was not a complete 3D model of this megasynthase. This study is the first to present a homology model for the whole hFAS, including its two mobile domains and possible poses of ACP throughout the cycle of fatty acid biosynthesis, thus mapping obligatory checkpoints in its trajectory. Hence, we believe that these structural insights will allow for future studies of the catalytic mechanism of the overall hFAS.


Assuntos
Ácido Graxo Sintase Tipo I/química , Animais , Domínio Catalítico , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Domínios Proteicos , Homologia de Sequência de Aminoácidos , Suínos
12.
Endocrinology ; 159(1): 557-569, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29077876

RESUMO

Increasing brown adipose tissue (BAT) activity is regarded as a potential treatment of obese, hyperglycemic patients with metabolic syndrome. Triiodothyronine (T3) is known to stimulate BAT activity by increasing mitochondrial uncoupling protein 1 (Ucp1) gene transcription, leading to increased thermogenesis and decreased body weight. Here we report our studies on the effects of T3 and glucose in two mouse models and in mouse immortalized brown preadipocytes in culture. We identified carbohydrate response element binding protein (ChREBP) as a T3 target gene in BAT by RNA sequencing and studied its effects in brown adipocytes. We found that ChREBP was upregulated by T3 in BAT in both hyperglycemic mouse models. In brown preadipocytes, T3 and glucose synergistically and dose dependently upregulated Ucp1 messenger RNA 1000-fold compared with low glucose concentrations. Additionally, we observed increased ChREBP and Ucp1 protein 11.7- and 19.9-fold, respectively, along with concomitant induction of a hypermetabolic state. Moreover, downregulation of ChREBP inhibited T3 and glucose upregulation of Ucp1 100-fold, whereas overexpression of ChREBP upregulated Ucp1 5.2-fold. We conclude that T3 and glucose signaling pathways coordinately regulate the metabolic state of BAT and suggest that ChREBP is a target for therapeutic regulation of BAT activity.


Assuntos
Adipócitos Marrons/metabolismo , Hiperglicemia/metabolismo , Proteínas Nucleares/metabolismo , Obesidade/metabolismo , Fatores de Transcrição/metabolismo , Tri-Iodotironina/metabolismo , Proteína Desacopladora 1/agonistas , Regulação para Cima , Transporte Ativo do Núcleo Celular , Adipócitos Marrons/citologia , Adipócitos Marrons/patologia , Adipogenia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Linhagem Celular Transformada , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Metabolismo Energético , Ácido Graxo Sintase Tipo I/química , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Perfilação da Expressão Gênica , Ontologia Genética , Transportador de Glucose Tipo 4/agonistas , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Hiperglicemia/etiologia , Hiperglicemia/patologia , Masculino , Camundongos Endogâmicos C57BL , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Obesidade/etiologia , Obesidade/patologia , Regiões Promotoras Genéticas , Interferência de RNA , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Tri-Iodotironina/administração & dosagem , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo
13.
J Biomol Struct Dyn ; 36(15): 4114-4125, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29161996

RESUMO

Thioesterase (TE) domain of fatty acid synthase (FAS) is an attractive therapeutic target for design and development of anticancer drugs. In this present work, we search for the potential FAS inhibitors of TE domain from the ZINC database based on similarity search using three natural compounds as templates, including flavonoids, terpenoids, and phenylpropanoids. Molecular docking was used to predict the interaction energy of each screened ligand compared to the reference compound, which is methyl γ-linolenylfluorophosphonate (MGLFP). Based on this computational technique, rosmarinic acid and its eight analogs were observed as a new series of potential FAS inhibitors, which showed a stronger binding affinity than MGLFP. Afterward, nine docked complexes were studied by molecular dynamics simulations for investigating protein-ligand interactions and binding free energies using MM-PB(GB)SA, MM-3DRISM-KH, and QM/MM-GBSA methods. The binding free energy calculation indicated that the ZINC85948835 (R34) displayed the strongest binding efficiency against the TE domain of FAS. There are eight residues (S2308, I2250, E2251, Y2347, Y2351, F2370, L2427, and E2431) mainly contributed for the R34 binding. Moreover, R34 could directly form hydrogen bonds with S2308, which is one of the catalytic triad of TE domain. Therefore, our finding suggested that R34 could be a potential candidate as a novel FAS-TE inhibitor for further drug design.


Assuntos
Antineoplásicos/química , Produtos Biológicos/química , Cinamatos/química , Depsídeos/química , Inibidores Enzimáticos/química , Ácido Graxo Sintase Tipo I/química , Simulação de Dinâmica Molecular , Domínio Catalítico , Cinamatos/farmacologia , Bases de Dados de Compostos Químicos , Depsídeos/farmacologia , Desenho de Fármacos , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Flavonoides/química , Ensaios de Triagem em Larga Escala , Humanos , Ligação de Hidrogênio , Cinética , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Terpenos/química , Termodinâmica , Ácido Rosmarínico
14.
Nat Chem Biol ; 13(4): 360-362, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28218911

RESUMO

Fungal type I fatty acid synthases (FASs) are mega-enzymes with two separated, identical compartments, in which the acyl carrier protein (ACP) domains shuttle substrates to catalytically active sites embedded in the chamber wall. We devised synthetic FASs by integrating heterologous enzymes into the reaction chambers and demonstrated their capability to convert acyl-ACP or acyl-CoA from canonical fatty acid biosynthesis to short/medium-chain fatty acids and methyl ketones.


Assuntos
Basidiomycota/enzimologia , Ácido Graxo Sintase Tipo I/metabolismo , Ácidos Graxos/metabolismo , Cetonas/metabolismo , Saccharomyces cerevisiae/enzimologia , Biocatálise , Ácido Graxo Sintase Tipo I/química , Ácidos Graxos/química , Cetonas/química , Modelos Moleculares , Estrutura Molecular
15.
J Biomol Struct Dyn ; 35(6): 1350-1366, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27145135

RESUMO

Human fatty acid synthase (hFASN), a homo dimeric lipogenic enzyme with seven catalytic domains, is an important clinical target in cancer, metabolic syndrome and infections. Here, molecular modelling and docking methods were implemented to examine the inter-molecular interactions of thioesterase (TE) domain in hFASN with its physiological substrate, and to identify potential chemical inhibitors. TE catalyses the hydrolysis of thioester bond between palmitate and the 4' phosphopantetheine of acyl carrier protein, releasing 16-carbon palmitate. The crystal structure of hFASN TE in two inhibitory conformations (A and B) were geometry-optimized and used for molecular docking with palmitate, orlistat (a known FASN inhibitor) and virtual screening against compounds from National Cancer Institute (NCI) database. Relatively, low binding affinity was observed during the complex formation of palmitate with A (-.164 kcal/mol) and B (-.332 kcal/mol) forms of TE, when compared with orlistat-docked TE (A form: -5.872 kcal/mol and B form: -5.484 kcal/mol), clearly indicating that the native inhibited conformation (crystal structure) was unfavourable for substrate binding. We used these orlistat dual binding modes as positive controls for prioritizing the ligands during virtual screening. From 2, 31,617 molecules in the NCI database, 916 high-scoring compounds (hit ligands) were obtained for A-form and 4582 for B-form of the TE-domain, which were then ranked according to glide docking score, XP H bond score, absorption, distribution, metabolism and excretion and binding free energy (Prime/MM-GBSA). Consequently, two top scoring ligands (NSC: 319661 and NSC: 153166) emerged as promising drug candidates that may be tested in FASN-over-expressing diseases.


Assuntos
Sítios de Ligação , Ácido Graxo Sintase Tipo I/química , Ligantes , Conformação Molecular , Simulação de Acoplamento Molecular , Domínios e Motivos de Interação entre Proteínas , Simulação por Computador , Desenho de Fármacos , Humanos , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Ligação Proteica
16.
J Biol Chem ; 291(7): 3520-30, 2016 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-26663084

RESUMO

The type I fatty acid synthase (FASN) is responsible for the de novo synthesis of palmitate. Chain length selection and release is performed by the C-terminal thioesterase domain (TE1). FASN expression is up-regulated in cancer, and its activity levels are controlled by gene dosage and transcriptional and post-translational mechanisms. In addition, the chain length of fatty acids produced by FASN is controlled by a type II thioesterase called TE2 (E.C. 3.1.2.14). TE2 has been implicated in breast cancer and generates a broad lipid distribution within milk. The molecular basis for the ability of the TE2 to compete with TE1 for the acyl chain attached to the acyl carrier protein (ACP) domain of FASN is unknown. Herein, we show that human TE1 efficiently hydrolyzes acyl-CoA substrate mimetics. In contrast, TE2 prefers an engineered human acyl-ACP substrate and readily releases short chain fatty acids from full-length FASN during turnover. The 2.8 Å crystal structure of TE2 reveals a novel capping domain insert within the α/ß hydrolase core. This domain is reminiscent of capping domains of type II thioesterases involved in polyketide synthesis. The structure also reveals that the capping domain had collapsed onto the active site containing the Ser-101-His-237-Asp-212 catalytic triad. This observation suggests that the capping domain opens to enable the ACP domain to dock and to place the acyl chain and 4'-phosphopantetheinyl-linker arm correctly for catalysis. Thus, the ability of TE2 to prematurely release fatty acids from FASN parallels the role of editing thioesterases involved in polyketide and non-ribosomal peptide synthase synthases.


Assuntos
Acil Coenzima A/metabolismo , Ácido Graxo Sintase Tipo I/metabolismo , Modelos Moleculares , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/genética , Proteína de Transporte de Acila/metabolismo , Acil Coenzima A/química , Sítios de Ligação , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Ácido Graxo Sintase Tipo I/química , Ácidos Graxos Voláteis/química , Ácidos Graxos Voláteis/metabolismo , Humanos , Hidrólise , Peso Molecular , Palmitoil Coenzima A/química , Palmitoil Coenzima A/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato
17.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 11): 1401-7, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26527268

RESUMO

While a deep understanding of the fungal and mammalian multi-enzyme type I fatty-acid synthases (FAS I) has been achieved in recent years, the bacterial FAS I family, which is narrowly distributed within the Actinomycetales genera Mycobacterium, Corynebacterium and Nocardia, is still poorly understood. This is of particular relevance for two reasons: (i) although homologous to fungal FAS I, cryo-electron microscopic studies have shown that bacterial FAS I has unique structural and functional properties, and (ii) M. tuberculosis FAS I is a drug target for the therapeutic treatment of tuberculosis (TB) and therefore is of extraordinary importance as a drug target. Crystals of FAS I from C. efficiens, a homologue of M. tuberculosis FAS I, were produced and diffracted X-rays to about 4.5 Å resolution.


Assuntos
Corynebacterium/enzimologia , Ácido Graxo Sintase Tipo I/química , Cristalização , Ácido Graxo Sintase Tipo I/isolamento & purificação , Mycobacterium tuberculosis/enzimologia , Difração de Raios X
18.
J Nutr ; 145(10): 2273-9, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26246323

RESUMO

BACKGROUND: Fructose induces nonalcoholic fatty liver disease (NAFLD). Citrulline (Cit) may exert a beneficial effect on steatosis. OBJECTIVE: We compared the effects of Cit and an isonitrogenous mixture of nonessential amino acids (NEAAs) on fructose-induced NAFLD. METHODS: Twenty-two male Sprague Dawley rats were randomly assigned into 4 groups (n = 4-6) to receive for 8 wk a 60% fructose diet, either alone or supplemented with Cit (1 g · kg(-1) · d(-1)), or an isonitrogenous amount of NEAAs, or the same NEAA-supplemented diet with starch and maltodextrin instead of fructose (controls). Nutritional and metabolic status, liver function, and expression of genes of hepatic lipid metabolism were determined. RESULTS: Compared with controls, fructose led to NAFLD with significantly higher visceral fat mass (128%), lower lean body mass (-7%), insulin resistance (135%), increased plasma triglycerides (TGs; 67%), and altered plasma amino acid concentrations with decreased Arg bioavailability (-27%). This was corrected by both NEAA and Cit supplementation. Fructose caused a 2-fold increase in the gene expression of fatty acid synthase (Fas) and 70% and 90% decreases in that of carnitine palmitoyl-transferase 1a and microsomal TG transfer protein via a nearly 10-fold higher gene expression of sterol regulatory element-binding protein-1c (Srebp1c) and carbohydrate-responsive element-binding protein (Chrebp), and a 90% lower gene expression of peroxisome proliferator-activated receptor α (Ppara). NEAA or Cit supplementation led to a Ppara gene expression similar to controls and decreased those of Srebp1c and Chrebp in the liver by 50-60%. Only Cit led to Fas gene expression and Arg bioavailability similar to controls. CONCLUSION: In our rat model, Cit and NEAAs effectively prevented fructose-induced NAFLD. On the basis of literature data and our findings, we propose that NEAAs may exert their effects specifically on the liver, whereas Cit presumably acts at both the hepatic and whole-body level, in part via improved peripheral Arg metabolism.


Assuntos
Aminoácidos/uso terapêutico , Citrulina/uso terapêutico , Suplementos Nutricionais , Ácido Graxo Sintase Tipo I/metabolismo , Fígado/enzimologia , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Algoritmos , Aminoácidos/sangue , Animais , Arginina/sangue , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/agonistas , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Biomarcadores/sangue , Ácido Graxo Sintase Tipo I/química , Ácido Graxo Sintase Tipo I/genética , Frutose/efeitos adversos , Frutose/antagonistas & inibidores , Regulação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Resistência à Insulina , Fígado/metabolismo , Fígado/fisiopatologia , Masculino , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Ornitina/sangue , PPAR alfa/agonistas , PPAR alfa/antagonistas & inibidores , PPAR alfa/genética , PPAR alfa/metabolismo , Distribuição Aleatória , Ratos Sprague-Dawley , Proteína de Ligação a Elemento Regulador de Esterol 1/agonistas , Proteína de Ligação a Elemento Regulador de Esterol 1/antagonistas & inibidores , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
19.
Biochemistry ; 54(13): 2205-13, 2015 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-25774789

RESUMO

Acyl carrier protein (ACP) domains are critical integral components of multifunctional type I fatty acid synthases (FAS I) and polyketide synthases (PKSs), where they shuttle the growing adducts of the synthesis between the catalytic domains. In contrast to ACP of mammalian FAS I, PKSs, and the dissociated fatty acid synthase type II systems (FAS II) of bacteria, fungal FAS I ACP consists of two subdomains, one comprising the canonical ACP fold observed in all FAS systems and the other representing an extra structural subdomain. While ACPs of dissociated FAS II are able to sequester the reaction intermediates during substrate shuttling, such a transport mechanism has not been observed in ACP domains of multifunctional FAS I and PKS systems. For a better understanding of the interaction between the canonical subdomain of fungal ACP with the growing acyl chain and the role of the structural subdomain, we determined the structure of the isolated Saccharomyces cerevisiae acyl carrier protein (ScACP) domain by NMR spectroscopy and investigated the interactions between ScACP and covalently attached substrate acyl chains of varying length by monitoring chemical shift perturbations. The interactions were mapped to the hydrophobic core of the canonical subdomain, while no perturbations were detected in the structural subdomain. A population analysis revealed that only approximately 15% of covalently attached decanoyl chains are sequestered by the ACP core, comparable to the mammalian FAS I and multifunctional PKS systems, which do not sequester their substrates. Finally, denaturation experiments show that both ScACP subdomains unfold cooperatively and that the weak interaction of the acyl chain with the hydrophobic core does not significantly affect the ACP stability.


Assuntos
Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/metabolismo , Ácido Graxo Sintase Tipo II/química , Ácido Graxo Sintase Tipo I/química , Proteínas de Saccharomyces cerevisiae/química , Ácido Graxo Sintase Tipo I/metabolismo , Ácido Graxo Sintase Tipo II/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Biochem J ; 459(3): 489-503, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24511897

RESUMO

VPA (valproic acid), a short-chain fatty acid that is a HDAC (histone deacetylase) inhibitor, is known to suppress adipogenesis. In the present study, we identified the molecular mechanism of VPA-mediated suppression of adipogenesis in adipocytes. VPA suppressed the accumulation of intracellular triacylglycerol. The expression levels of PPARγ (peroxisome-proliferator-activated receptor γ) and C/EBPα (CCAAT/enhancer-binding protein α), which are key regulators of adipogenesis, as well as the expression of SCD (stearoyl-CoA desaturase), were decreased by the treatment with VPA. Moreover, glycerol release was decreased in the VPA-treated cells, even though the transcription levels of ATGL (adipose triacylglycerol lipase), HSL (hormone-sensitive lipase) and MGL (monoacylglycerol lipase), all of which are involved in lipolysis, were elevated by the treatment with VPA. It is noteworthy that the expression level of FAS (fatty acid synthase) was significantly suppressed when the cells were cultured in medium containing VPA. Furthermore, VPA-mediated suppression of the accumulation of the intracellular triacylglycerols was prevented by the treatment with palmitic acid, a major product of FAS. The results of promoter-luciferase and chromatin immunoprecipitation assays demonstrated that USF1(upstream stimulating factor 1) bound to the E-box of the promoter region of the FAS gene. In addition, the expression of USF1 was decreased by the treatment with VPA. siRNA-mediated knockdown of the expression of the USF1 gene repressed adipogenesis along with the decreased expression of the FAS gene. The overexpression of USF1 enhanced both adipogenesis and the expression of FAS in VPA-treated cells. These results indicate that VPA suppressed adipogenesis through the down-regulation of USF1-activated fatty acid synthesis in adipocytes.


Assuntos
Adipócitos Brancos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Fatores Estimuladores Upstream/antagonistas & inibidores , Ácido Valproico/farmacologia , Células 3T3-L1 , Adipócitos Brancos/citologia , Adipócitos Brancos/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/antagonistas & inibidores , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Ácido Graxo Sintase Tipo I/química , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Ácidos Graxos/biossíntese , Ácidos Graxos não Esterificados/metabolismo , Genes Reporter/efeitos dos fármacos , Camundongos , PPAR gama/antagonistas & inibidores , PPAR gama/genética , PPAR gama/metabolismo , Ácido Palmítico/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estearoil-CoA Dessaturase/antagonistas & inibidores , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Triglicerídeos/biossíntese , Fatores Estimuladores Upstream/genética , Fatores Estimuladores Upstream/metabolismo , Ácido Valproico/antagonistas & inibidores
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