RESUMEN
Sterols in eukaryotic cells play important roles in modulating membrane fluidity and in cell signaling and trafficking. During evolution, a combination of gene losses and acquisitions gave rise to an extraordinary diversity of sterols in different organisms. The sterol C-22 desaturase identified in plants and fungi as a cytochrome P-450 monooxygenase evolved from the first eukaryotic cytochrome P450 and was lost in many lineages. Although the ciliate Tetrahymena thermophila desaturates sterols at the C-22 position, no cytochrome P-450 orthologs are present in the genome. Here, we aim to identify the genes responsible for the desaturation as well as their probable origin. We used gene knockout and yeast heterologous expression approaches to identify two putative genes, retrieved from a previous transcriptomic analysis, as sterol C-22 desaturases. Furthermore, we demonstrate using bioinformatics and evolutionary analyses that both genes encode a novel type of sterol C-22 desaturase that belongs to the large fatty acid hydroxylase/desaturase superfamily and the genes originated by genetic duplication prior to functional diversification. These results stress the widespread existence of nonhomologous isofunctional enzymes among different lineages of the tree of life as well as the suitability for the use of T. thermophila as a valuable model to investigate the evolutionary process of large enzyme families.
Asunto(s)
Proteínas Protozoarias , Estearoil-CoA Desaturasa , Tetrahymena thermophila , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Saccharomyces cerevisiae , Estearoil-CoA Desaturasa/química , Estearoil-CoA Desaturasa/clasificación , Estearoil-CoA Desaturasa/genética , Esteroles/metabolismo , Tetrahymena thermophila/enzimología , Filogenia , Proteínas Protozoarias/química , Proteínas Protozoarias/clasificación , Proteínas Protozoarias/genéticaRESUMEN
Stearoyl-CoA desaturase-1 (SCD1 or delta-9 desaturase, D9D) is a key metabolic protein that modulates cellular inflammation and stress, but overactivity of SCD1 is associated with diseases, including cancer and metabolic syndrome. This transmembrane endoplasmic reticulum protein converts saturated fatty acids into monounsaturated fatty acids, primarily stearoyl-CoA into oleoyl-CoA, which are critical products for energy metabolism and membrane composition. The present computational molecular dynamics study characterizes the molecular dynamics of SCD1 with substrate, product, and as an apoprotein. The modeling of SCD1:fatty acid interactions suggests that: (1) SCD1:CoA moiety interactions open the substrate-binding tunnel, (2) SCD1 stabilizes a substrate conformation favorable for desaturation, and (3) SCD1:product interactions result in an opening of the tunnel, possibly allowing product exit into the surrounding membrane. Together, these results describe a highly dynamic series of SCD1 conformations resulting from the enzyme:cofactor:substrate interplay that inform drug-discovery efforts.
Asunto(s)
Simulación por Computador , Estearoil-CoA Desaturasa/metabolismo , Apoproteínas/metabolismo , Coenzima A/metabolismo , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Unión Proteica , Estearoil-CoA Desaturasa/química , Especificidad por Sustrato , TermodinámicaRESUMEN
A full understanding of the catalytic action of non-heme iron (NHFe) and non-heme diiron (NHFe2) enzymes is still beyond the grasp of contemporary computational and experimental techniques. Many of these enzymes exhibit fascinating chemo-, regio-, and stereoselectivity, in spite of employing highly reactive intermediates which are necessary for activations of most stable chemical bonds. Herein, we study in detail one intriguing representative of the NHFe2 family of enzymes: soluble Δ9 desaturase (Δ9D), which desaturates rather than performing the thermodynamically favorable hydroxylation of substrate. Its catalytic mechanism has been explored in great detail by using QM(DFT)/MM and multireference wave function methods. Starting from the spectroscopically observed 1,2-µ-peroxo diferric P intermediate, the proton-electron uptake by P is the favored mechanism for catalytic activation, since it allows a significant reduction of the barrier of the initial (and rate-determining) H-atom abstraction from the stearoyl substrate as compared to the "proton-only activated" pathway. Also, we ruled out that a Q-like intermediate (high-valent diamond-core bis-µ-oxo-[FeIV]2 unit) is involved in the reaction mechanism. Our mechanistic picture is consistent with the experimental data available for Δ9D and satisfies fairly stringent conditions required by Nature: the chemo-, stereo-, and regioselectivity of the desaturation of stearic acid. Finally, the mechanisms evaluated are placed into a broader context of NHFe2 chemistry, provided by an amino acid sequence analysis through the families of the NHFe2 enzymes. Our study thus represents an important contribution toward understanding the catalytic action of the NHFe2 enzymes and may inspire further work in NHFe(2) biomimetic chemistry.
Asunto(s)
Electrones , Protones , Estearoil-CoA Desaturasa/metabolismo , Sitios de Unión , Biocatálisis , Teoría Funcional de la Densidad , Modelos Moleculares , Solubilidad , Estearoil-CoA Desaturasa/químicaRESUMEN
Stearoyl-CoA desaturase 1 (SCD1) is a membrane-embedded metalloenzyme that catalyzes the formation of a double bond on a saturated acyl-CoA. SCD1 has a diiron center and its proper function requires an electron transport chain composed of NADH (or NADPH), cytochrome b5 reductase (b5R), and cytochrome b5 (cyt b5). Since SCD1 is a key regulator in fat metabolism and is required for survival of cancer cells, there is intense interest in targeting SCD1 for various metabolic diseases and cancers. Crystal structures of human and mouse SCD1 were reported recently; however, both proteins have two zinc ions instead of two iron ions in the catalytic center, and as a result, the enzymes are inactive. Here we report a general approach for incorporating iron into heterologously expressed proteins in HEK293 cells. We produced mouse SCD1 that contains a diiron center and visualized its diiron center by solving its crystal structure to 3.5 Å. We assembled the entire electron transport chain using the purified soluble domains of cyt b5 and b5R, and the purified mouse SCD1, and we showed that three proteins coordinate to produce proper products. These results established an in vitro system that allows precise perturbations of the electron transport chain for the understanding of the catalytic mechanism in SCD1.
Asunto(s)
Citocromo-B(5) Reductasa/metabolismo , Citocromos b5/metabolismo , Estearoil-CoA Desaturasa/química , Estearoil-CoA Desaturasa/metabolismo , Animales , Dominio Catalítico , Cristalografía por Rayos X , Células HEK293 , Humanos , Hierro/metabolismo , Ratones , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Células Sf9 , Estearoil-CoA Desaturasa/genética , Zinc/metabolismoRESUMEN
Stearyl coenzyme A desaturase enzyme 1 (SCD1) is a key enzyme that catalyzes the conversion of saturated fatty acids (SFA) into monounsaturated fatty acids (MUFA) and plays a vital role in lipid metabolism of tumor cells. SCD1 is overexpressed in a variety of malignant tumors, and its related inhibitors showed significant anti-tumor activity in vitro and in vivo experiments, which is a new target for tumor therapy. The focus of this study is to identify novel SCD1 inhibitors from natural products through computer simulations. First, 176,602 compounds from natural product databases were virtually screened. By molecular dynamics (MD) simulations, the ligand-protein interactions of 5 compounds with high docking manifestation were analyzed accurately. Then, MM-GBSA and MM-PBMA methods were used to verify the results. Finally, ADMET prediction was performed for the 5 compounds. As a result, two natural products with potential inhibition towards SCD1 were identified, which had the excellent docking manifestation, binding mode within SCD1 pocket and stability during molecular dynamics simulation. This study provides a meaningful model for the development and optimization of new inhibitors and anti-tumor drugs targeting SCD1.
Asunto(s)
Antineoplásicos/química , Productos Biológicos/química , Estearoil-CoA Desaturasa/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Productos Biológicos/farmacología , Proteínas Sanguíneas/metabolismo , Barrera Hematoencefálica/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Descubrimiento de Drogas , Humanos , Absorción Intestinal , Dosificación Letal Mediana , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Pruebas de Mutagenicidad , Ratas , Estearoil-CoA Desaturasa/químicaRESUMEN
Δ9-Fatty acid desaturase (Δ9-desaturase) is a rate-limiting enzyme of unsaturated fatty acid biosynthesis in animal cells and specifically introduces a cis-double bond at the Δ9-position of acyl-CoA. Since the chemical structure of fatty acids determines the physicochemical properties of cellular membrane and modulates a broad range of cellular functions, double bond introduction into a fatty acid by Δ9-desaturase should be specifically carried out. Reported crystal structures of stearoyl-CoA desaturase (SCD)1, one of the most studied Δ9-desaturases, have revealed the mechanism underlying the determination of substrate preference, as well as the position (Δ9) and conformation (cis) of double bond introduction. The crystal structures of SCD1 have also provided insights into the function of other Δ9-desaturases, including Drosophila homologs. Moreover, the amino-terminal sequences of Δ9-desaturases are shown to have unique roles in protein degradation. In this review, we introduce recent advances in the understanding of the function and regulation of Δ9-desaturase from the standpoint of protein structure.
Asunto(s)
Ácido Graso Desaturasas/química , Secuencia de Aminoácidos , Animales , Ácido Graso Desaturasas/metabolismo , Ácidos Grasos/biosíntesis , Humanos , Estructura Terciaria de Proteína , Alineación de Secuencia , Estearoil-CoA Desaturasa/química , Estearoil-CoA Desaturasa/metabolismo , Especificidad por SustratoRESUMEN
Pseudomonas putida counteract the fluidizing effect of cationic surfactants decreasing the content of membrane unsaturated fatty acid (UFA). A Δ9-fatty acid desaturase gene (desA) from P. putida was isolated, cloned, and successfully expressed in Escherichia coli, a Δ9 desaturase deficient organism. desA consists of 1185 bp and codes for 394 amino acids. The deduced amino acid sequence reveals three histidine clusters and a hydropathy profile, typical of membrane-bound desaturases. Validating desA expression in E. coli cells, the amount of palmitoleic acid increased from 2.05 to 7.36%, with the concomitant increase in membrane fluidity (fluorescence polarization value decrease from 0.13 ± 0.03 to 0.09 ± 0.02). Also, when DesA activity was assayed in vivo, the percentage of UFA obtained from exogenous palmitic acid [1-14 C] increased 10-fold. In contrast, when cells expressing desA were exposed 15 min at sublethal concentration of cationic surfactants, the amount of UFA was 82% lower than that detected in cells non-exposed. Thus, the decrease in UFA content to counteract the fluidizing effect of cationic surfactants can be correlated with reduction of DesA activity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ácidos Grasos Insaturados/metabolismo , Pseudomonas putida/enzimología , Estearoil-CoA Desaturasa/metabolismo , Tensoactivos/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Expresión Génica , Fluidez de la Membrana/efectos de los fármacos , Pseudomonas putida/efectos de los fármacos , Pseudomonas putida/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estearoil-CoA Desaturasa/química , Estearoil-CoA Desaturasa/genética , Tensoactivos/farmacologíaRESUMEN
Stearoyl Co A desaturase (SCD) is a rate-limiting lipogenic enzyme that plays an integral role in catalyzing the synthesis of monounsaturated fatty acids, chiefly oleate and palmitoleate. Both contribute a major part of the biological membrane. Numerous SCD isoforms exist in mouse and humans, i.e., SCD-1 to SCD-4 and SCD-1 and SCD-5, respectively. From the biological viewpoint, hyperexpression of SCD1 cause many metabolic disorders including obesity, insulin resistance, hypertension, and hypertriglyceridemia, etc. Herein, an effort has been made to highlight the value of protein engineering in controlling the SCD-1 expression with the involvement of different inhibitors as therapeutic agents. The first part of the review describes Stearoyl CoA desaturase index and different SCD isoforms. Various regulatory aspects of SCD are reviewed in four subsections, i.e., (1) hormonal regulation, (2) regulation by dietary carbohydrates, (3) regulation by green tea, and (4) regulation via polyunsaturated fatty acids (PUFAs). Moreover, the regulation of Stearoyl CoA desaturase expression in the metabolism of fats and carbohydrates is discussed. The third part mainly focuses on natural and synthetic inhibitors. Towards the end, information is also given on potential future considerations of SCD-1 inhibitors as metabolic syndrome therapeutics, yet additional work is required.
Asunto(s)
Inhibidores Enzimáticos , Regulación Enzimológica de la Expresión Génica , Síndrome Metabólico , Ingeniería de Proteínas/métodos , Estearoil-CoA Desaturasa , Animales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/biosíntesis , Isoenzimas/química , Isoenzimas/genética , Síndrome Metabólico/tratamiento farmacológico , Síndrome Metabólico/enzimología , Síndrome Metabólico/genética , Estearoil-CoA Desaturasa/antagonistas & inhibidores , Estearoil-CoA Desaturasa/biosíntesis , Estearoil-CoA Desaturasa/química , Estearoil-CoA Desaturasa/genéticaRESUMEN
PURPOSE: DNA methylation is one of the most extensively studied mechanisms within epigenetics, and it is suggested that diet-induced changes in methylation status could be involved in energy metabolism regulation. Conjugated linoleic acid (CLA) and calcium supplementation counteract body weight gain, particularly under a high-fat (HF) diet, in adult mice. The aim was to determine whether the modulation of DNA methylation pattern in target genes and tissues could be an underlying mechanism of action. METHODS: Mice (C57BL/6J) were divided into five groups according to diet and treatment: normal fat as the control group (12 % kJ content as fat), HF group (43 % kJ content as fat), HF + CLA (6 mg CLA/day), HF + calcium (12 g/kg of calcium) and HF with both compounds. Gene expression and methylation degree of CpG sites in promoter sequences of genes involved in fatty acid metabolism, including adiponectin (Adipoq), stearoyl-CoA desaturase (Scd1) and fatty acid synthase (Fasn), were determined by bisulphite sequencing in liver and epididymal white adipose tissue. RESULTS: Results showed that the methylation profile of promoters was significantly altered by dietary supplementation in a gene- and tissue-specific manner, whereas only slight changes were observed in the HF group. Furthermore, changes in specific CpG sites were also associated with an overall healthier metabolic profile, in particular for calcium-receiving groups. CONCLUSIONS: Both CLA and calcium were able to modify the methylation pattern of genes involved in energy balance in adulthood, which opens a novel area for increasing efficiency in body weight management strategies.
Asunto(s)
Fármacos Antiobesidad/uso terapéutico , Calcio de la Dieta/uso terapéutico , Metilación de ADN , Suplementos Dietéticos , Epigénesis Genética , Ácidos Linoleicos Conjugados/uso terapéutico , Obesidad/prevención & control , Adiponectina/antagonistas & inhibidores , Adiponectina/genética , Adiponectina/metabolismo , Tejido Adiposo Blanco/enzimología , Tejido Adiposo Blanco/metabolismo , Animales , Islas de CpG , Dieta Alta en Grasa/efectos adversos , Metabolismo Energético , Acido Graso Sintasa Tipo I/antagonistas & inhibidores , Acido Graso Sintasa Tipo I/genética , Acido Graso Sintasa Tipo I/metabolismo , Hígado/enzimología , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Obesidad/etiología , Obesidad/metabolismo , Especificidad de Órganos , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Estearoil-CoA Desaturasa/química , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismo , Aumento de PesoRESUMEN
CD14 is a co-receptor involved in the recognition of Gram-negative and Gram-positive bacteria, the latter known to cause dental caries. The aim of this study was to determine whether soluble CD14 (sCD14) in saliva was associated with caries activity and the collection method from the saliva. The study included 55 participants aged 20 to 40 years, 30 with dental caries and 25 caries-free controls. We collected 110 saliva samples in total, 55 of resting saliva and 55 of mechanically stimulated saliva. Median levels of sCD14, measured with a matrix-matched enzyme-linked immunosorbent assay (ELISA), were higher in the caries-active than in the caries-free group in either resting (203.3 vs.167.9 ng mL-1; P<0.01) or stimulated saliva (201 vs.105.7 ng mL-1; P<0.01). The resting salivary flow rate was lower in the caries-active than caries-free group (0.61±0.42 vs. 0.98±0.52 mL min-1; P<0.01). Hyposalivation was observed only in the caries-active group (10 and 13 % in stimulated and resting saliva, respectively). Higher salivary sCD14 levels and secretion rates were clearly associated with dental caries and resting saliva. Future studies should focus on the clinical utility of salivary sCD14 as a potential biomarker and predictor of future caries events.
Asunto(s)
Biomarcadores/análisis , Biomarcadores/química , Caries Dental/inducido químicamente , Saliva/química , Estearoil-CoA Desaturasa/análisis , Estearoil-CoA Desaturasa/química , Adulto , Croacia , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Adulto JovenRESUMEN
Stearoyl-CoA desaturase (SCD), the central enzyme in the biosynthesis of monounsaturated fatty acids, introduces a cis-Δ9 double bond into saturated fatty acids. SCD-1 has been proposed as promising target for the treatment of cancer, skin disorders and metabolic diseases, and strong efforts have been made during the last decade to develop clinical drug candidates. While the regulation and biological implications of SCD-1 have been extensively reviewed, the molecular mechanisms through which SCD-1 mediates cellular responses remained a mystery. An important aspect seems to be that SCD-1 induces adaptive stress signaling that maintains cellular persistence and fosters survival and cellular functionality under distinct pathological conditions. Here, we will first provide an overview about the function, regulation, structure and mechanism of SCD-1 and then focus on mitogenic and stress-related signal transduction pathways orchestrated by SCD-1. Moreover, we will discuss molecular mechanisms and potential lipid factors that link SCD-1 activity with initial signal transduction.
Asunto(s)
Transducción de Señal , Estearoil-CoA Desaturasa/metabolismo , Estrés Fisiológico , Animales , Biocatálisis , Supervivencia Celular , Humanos , Modelos Biológicos , Estearoil-CoA Desaturasa/químicaRESUMEN
Tea (Camellia sinensis L.) is a thermophilic evergreen woody plant that has poor cold tolerance. The SAD gene plays a key role in regulating fatty acid synthesis and membrane lipid fluidity in response to temperature change. In this study, full-length SAD cDNA was cloned from tea leaves using rapid amplification of cDNA ends and polymerase chain reaction (PCR)-based methods. Sequence analysis demonstrated that CsSAD had a high similarity to other corresponding cDNAs. At 25°C, the CsSAD transcriptional level was highest in the leaf and lowest in the stem, but there was no obvious difference between the root and stem organs. CsSAD expression was investigated by reverse transcription-PCR, which showed that CsSAD was upregulated at 4° and -5°C. At 25°C, CsSAD was induced by polyethylene glycol, abscisic acid, and wounding, and a similar trend was observed at 4°C, but the mean expression level at 4°C was lower than that at 25°C. Under natural cold acclimation, the 'CsCr05' variety's CsSAD expression level increased before decreasing. The CsSAD expression level in variety 'CsCr06' showed no obvious change at first, but rapidly increased to a maximum when the temperature was very low. Our study demonstrates that CsSAD is upregulated in response to different abiotic conditions, and that it is important to study the stress resistance of the tea plant, particularly in response to low temperature, drought, and wounding.
Asunto(s)
Adaptación Fisiológica , Camellia sinensis/enzimología , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Estearoil-CoA Desaturasa/genética , Secuencia de Aminoácidos , Camellia sinensis/genética , Camellia sinensis/fisiología , Clonación Molecular , Frío , Sequías , Datos de Secuencia Molecular , Filogenia , Componentes Aéreos de las Plantas/enzimología , Componentes Aéreos de las Plantas/fisiología , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Raíces de Plantas/enzimología , Raíces de Plantas/fisiología , Alineación de Secuencia , Estearoil-CoA Desaturasa/química , Estearoil-CoA Desaturasa/metabolismoRESUMEN
The objective of this research was to study the delta-9 desaturation of individual trans (t) fatty acids that can be found in ruminant fat or partially hydrogenated vegetable oils (PHVO) and determine their effects on lipogenic gene expression in adipocytes. It was hypothesized that delta-9 desaturation and lipogenic properties of t-18:1 isomers depend on the position of double bond. Differentiated 3T3-L1 adipocytes were treated with 200 µM of t6-18:1, t9-18:1, t11-18:1, t13-18:1 or t16-18:1, cis (c)-9 18:1 or bovine serum albumin (BSA) vehicle control for 48 h. Cells were then harvested for fatty acid and gene expression analyses using gas chromatography and quantitative PCR respectively. Among t-18:1 isomers, t13-18:1 and t11-8:1 had the greatest percent delta-9 desaturation (44 and 41 % respectively) followed by t16-18:1 and t6-18:1 (32 and 17 % respectively), while c9-18:1 and t9-18:1 did not undergo delta-9 desaturation. Trans9-18:1 up-regulated (P < 0.05) the expression of lipogenic genes including fatty acid synthase and stearoyl-CoA desaturase-1 (P < 0.05), whereas the expression of these genes were not affected with other t-18:1 isomers (P > 0.05). Consistent with gene expression results, t9-18:1 increased the de novo lipogenic index (16:0/18:2n-6) compared with control cells and increased delta-9 desaturation index (c9-16:1/18:0) compared to other t-18:1 isomers (P < 0.05). The current study provides further evidence that the predominant trans fatty acid in PHVO (t9-18:1) has isomer specific lipogenic properties.
Asunto(s)
Adipocitos Blancos/enzimología , Ácido Graso Desaturasas/metabolismo , Ácido Graso Sintasas/metabolismo , Lipogénesis , Ácidos Oléicos/metabolismo , Estearoil-CoA Desaturasa/metabolismo , Ácidos Grasos trans/metabolismo , Células 3T3-L1 , Adipocitos Blancos/metabolismo , Animales , Supervivencia Celular , Grasas de la Dieta/análisis , Inducción Enzimática , Ácido Graso Sintasas/química , Ácido Graso Sintasas/genética , Alimentos en Conserva/análisis , Hidrogenación , Ratones , Estructura Molecular , Ácidos Oléicos/química , Aceites de Plantas/química , ARN Mensajero , Estearoil-CoA Desaturasa/química , Estearoil-CoA Desaturasa/genética , Estereoisomerismo , Ácidos Grasos trans/químicaRESUMEN
Berberine (BBR) shows very low plasma levels after oral administration due to its poor absorption by the gastrointestinal tract. We have previously demonstrated that BBR showed increased gastrointestinal absorption and enhanced antidiabetic effects in db/db mice after being entrapped into solid lipid nanoparticles (SLNs). However, whether BBR-loaded SLNs (BBR-SLNs) also have beneficial effects on hepatosteatosis is not clear. We investigated the effects of BBR-SLNs on lipid metabolism in the liver using histological staining and reverse transcription polymerase chain reaction analysis. The results showed that oral administration of BBR-SLNs inhibited the increase of body weight and decreased liver weight in parallel with the reduction of serum alanine transaminase and liver triglyceride levels in db/db mice. The maximum drug concentration in the liver was 20-fold higher than that in the blood. BBR-SLNs reduced fat accumulation and lipid droplet sizes significantly in the liver, as indicated by hematoxylin and eosin and Oil Red O staining. The expression of lipogenic genes, including fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD1), and sterol regulatory element-binding protein 1c (SREBP1c) were downregulated, while lipolytic gene carnitine palmitoyltransferase-1 (CPT1) was upregulated in BBR-SLN-treated livers. In summary, we have uncovered an unexpected effect of BBR-SLNs on hepatosteatosis treatment through the inhibition of lipogenesis and the induction of lipolysis in the liver of db/db mice.
Asunto(s)
Berberina/farmacología , Hígado Graso/tratamiento farmacológico , Lípidos/química , Hígado/efectos de los fármacos , Nanopartículas/química , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Berberina/sangre , Berberina/química , Peso Corporal/efectos de los fármacos , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Ácidos Grasos no Esterificados/sangre , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Tamaño de los Órganos/efectos de los fármacos , Estearoil-CoA Desaturasa/química , Estearoil-CoA Desaturasa/farmacología , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Triglicéridos/metabolismo , Regulación hacia ArribaRESUMEN
Stearoyl-coenzyme A desaturase-1 (SCD1) has an important role in lipid metabolism, and SCD1 inhibitors are potential therapeutic agents for the treatment of metabolic diseases and cancers. Here we report the 3.25-Å crystal structure of human SCD1 in complex with its substrate, stearoyl-coenzyme A, which defines the new SCD1 dimetal catalytic center and reveals the determinants of substrate binding to provide insights into the catalytic mechanism of desaturation of the stearoyl moiety. The structure also provides a mechanism for localization of SCD1 in the endoplasmic reticulum: human SCD1 folds around a tight hydrophobic core formed from four long α-helices that presumably function as an anchor spanning the endoplasmic reticulum membrane. Furthermore, our results provide a framework for the rational design of pharmacological inhibitors targeting the SCD1 enzyme.
Asunto(s)
Acilcoenzima A/metabolismo , Estearoil-CoA Desaturasa/química , Estearoil-CoA Desaturasa/metabolismo , Acilcoenzima A/química , Sitios de Unión , Cristalografía por Rayos X , Humanos , Metales/metabolismo , Modelos Moleculares , Conformación ProteicaRESUMEN
Stearoyl-CoA desaturase (SCD) is conserved in all eukaryotes and introduces the first double bond into saturated fatty acyl-CoAs. Because the monounsaturated products of SCD are key precursors of membrane phospholipids, cholesterol esters and triglycerides, SCD is pivotal in fatty acid metabolism. Humans have two SCD homologues (SCD1 and SCD5), while mice have four (SCD1-SCD4). SCD1-deficient mice do not become obese or diabetic when fed a high-fat diet because of improved lipid metabolic profiles and insulin sensitivity. Thus, SCD1 is a pharmacological target in the treatment of obesity, diabetes and other metabolic diseases. SCD1 is an integral membrane protein located in the endoplasmic reticulum, and catalyses the formation of a cis-double bond between the ninth and tenth carbons of stearoyl- or palmitoyl-CoA. The reaction requires molecular oxygen, which is activated by a di-iron centre, and cytochrome b5, which regenerates the di-iron centre. To understand better the structural basis of these characteristics of SCD function, here we crystallize and solve the structure of mouse SCD1 bound to stearoyl-CoA at 2.6 Å resolution. The structure shows a novel fold comprising four transmembrane helices capped by a cytosolic domain, and a plausible pathway for lateral substrate access and product egress. The acyl chain of the bound stearoyl-CoA is enclosed in a tunnel buried in the cytosolic domain, and the geometry of the tunnel and the conformation of the bound acyl chain provide a structural basis for the regioselectivity and stereospecificity of the desaturation reaction. The dimetal centre is coordinated by a unique spacial arrangement of nine conserved histidine residues that implies a potentially novel mechanism for oxygen activation. The structure also illustrates a possible route for electron transfer from cytochrome b5 to the di-iron centre.
Asunto(s)
Estearoil-CoA Desaturasa/química , Acilcoenzima A/química , Acilcoenzima A/metabolismo , Animales , Sitios de Unión , Cristalografía por Rayos X , Citocromos b5/química , Citocromos b5/metabolismo , Transporte de Electrón , Histidina/química , Histidina/metabolismo , Hierro/metabolismo , Ratones , Modelos Moleculares , Oxígeno/metabolismo , Estructura Terciaria de Proteína , Electricidad Estática , Estearoil-CoA Desaturasa/metabolismo , Relación Estructura-ActividadRESUMEN
Oleic (cis9-18:1), linoleic (cis9,cis12-18:2) and α-linolenic (cis9,cis12,cis15-18:3) acids are well described substrates of the Δ6-desaturase encoded by the mammalian fatty acid desaturase 2 (FADS2) gene. In addition, at least 9 other very structurally different fatty acids have been shown to be Δ6- or even Δ8-desaturated by the FADS2 protein. A better characterization of the substrate specificity of this enzyme is therefore needed. By using commercial cis9-18:1 and chemically synthesized cis12- and cis15-18:1 (sharing the n-6 double bond with 18:2 n-6 and the n-3 double bond with 18:3 n-3, respectively), we tried to decrypt the fatty acid structure driving the FADS2 substrate affinity. We first showed that both recombinant and native rat FADS2 were able to Δ6-desaturate not only the cis9- but also the cis12- and cis15-18:1 isomers. Next, the inhibitory effect of increasing concentrations of each 18:1 isomer was investigated in vitro on the Δ6-desaturation of α-linolenic acid. At equimolar inhibitor/substrate ratio (60 µM), the cis9-18:1 exhibited a significantly higher inhibition (25%) than the cis12- (8%) and cis15-18:1 (5%). This study shows that a single cis double bond in 12- or 15-position in 18:1 is enough to make them low Δ6-desaturable substrates. If a preexisting cis9-double bond is not absolutely required for the Δ6-desaturation of octadecenoic acids, its presence is however crucial to explain the higher enzyme affinity. Compared with oleic acid, the additional presence of a cis12-double bond in linoleic acid increased its inhibitory effect on the Δ6-desaturation of α-linolenic acid at low concentration (30 µM) but not at higher concentrations (60 and 120 µM). In this classification of the decreasing impact of the double bond when it comes closer to the methyl end of octadecenoic acids, the cis11-18:1 (cis-vaccenic acid) should be considered apart since it is itself not Δ6-desaturated but still a good competitive inhibitor of the α-linolenic acid Δ6-desaturation.
Asunto(s)
Ácidos Esteáricos/química , Ácidos Esteáricos/metabolismo , Estearoil-CoA Desaturasa/química , Estearoil-CoA Desaturasa/metabolismo , Animales , Sitios de Unión , Catálisis , Activación Enzimática , Isomerismo , Masculino , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The aim of the present research was to examine the effect of conjugated linoleic acids (CLA) supplementation on the activity of enzymes that take part in the synthesis of arachidonic acid (AA) and also to investigate the relation between their activity and the neoplastic process. The enzyme activities were established indirectly, because their measure was the amount of AA formed in vitro, being developed from linoleic acid as determined in liver microsomes of Spraque-Dawley rats. In addition, the indices of Δ6-desaturase (D6D) and Δ5-desaturase (D5D) were determined. To this aim, the method of high per-formance liquid chromatography with UV/VIS detection was used. Between the examined groups, statistically significant differences were observed in the activities of enzymes as well as D6D. The carcinogenic agent applied (DMBA) was found to significantly increase the activity of the examined enzymes. Negative correlation was found between the activities of desaturases and CLA supplementation, whereas the activity of those enzymes was a little higher in the group of rats with chemically induced cancer process. The neoplastic process has a stimulating effect on the activity of D6D. The decrease of D6D activity, resulting from the presence of CLA in the animals' diet, may confirm the anticancer properties of these isomers.
Asunto(s)
Anticarcinógenos/uso terapéutico , Ácido Araquidónico/metabolismo , Suplementos Dietéticos , Ácidos Linoleicos Conjugados/uso terapéutico , Hígado/enzimología , Neoplasias Mamarias Experimentales/prevención & control , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenocarcinoma/prevención & control , Animales , Anticarcinógenos/administración & dosificación , delta-5 Desaturasa de Ácido Graso , Ácido Graso Desaturasas/química , Ácido Graso Desaturasas/metabolismo , Ácidos Linoleicos Conjugados/administración & dosificación , Hígado/metabolismo , Hígado/patología , Masculino , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Tamaño de los Órganos , Distribución Aleatoria , Ratas Sprague-Dawley , Estearoil-CoA Desaturasa/química , Estearoil-CoA Desaturasa/metabolismo , Análisis de Supervivencia , Carga TumoralRESUMEN
The results of density matrix renormalization group complete active space self-consistent field (DMRG-CASSCF) and second-order perturbation theory (DMRG-CASPT2) calculations are presented on various structural alternatives for the O-O and first C-H activating step of the catalytic cycle of the binuclear nonheme iron enzyme Δ(9) desaturase. This enzyme is capable of inserting a double bond into an alkyl chain by double hydrogen (H) atom abstraction using molecular O2. The reaction step studied here is presumably associated with the highest activation barrier along the full pathway; therefore, its quantitative assessment is of key importance to the understanding of the catalysis. The DMRG approach allows unprecedentedly large active spaces for the explicit correlation of electrons in the large part of the chemically important valence space, which is apparently conditio sine qua non for obtaining well-converged reaction energetics. The derived reaction mechanism involves protonation of the previously characterized 1,2-µ peroxy Fe(III)Fe(III) (P) intermediate to a 1,1-µ hydroperoxy species, which abstracts an H atom from the C10 site of the substrate. An Fe(IV)-oxo unit is generated concomitantly, supposedly capable of the second H atom abstraction from C9. In addition, several popular DFT functionals were compared to the computed DMRG-CASPT2 data. Notably, many of these show a preference for heterolytic C-H cleavage, erroneously predicting substrate hydroxylation. This study shows that, despite its limitations, DMRG-CASPT2 is a significant methodological advancement toward the accurate computational treatment of complex bioinorganic systems, such as those with the highly open-shell diiron active sites.
Asunto(s)
Dominio Catalítico , Hierro/metabolismo , Teoría Cuántica , Estearoil-CoA Desaturasa/química , Estearoil-CoA Desaturasa/metabolismo , Modelos Moleculares , TermodinámicaRESUMEN
Desaturases catalyse modifications to fatty acids which are essential to homeostasis and for pheromone and defensive chemical production. All desaturases of the flour beetle Tribolium castaneum were investigated via query of the sequenced genome which yielded 15 putative acyl-Coenzyme A genes. Eleven desaturase mRNA were obtained in full length and functionally expressed in yeast. Phylogenetic analysis separated the desaturases into 4 distinct clades; one clade contained conserved beetle Δ9 desaturases, second clade was Tribolium-specific having diverse activities including Δ5, Δ9 and Δ12 desaturation and the other 2 clades had mixed insect representatives. Three members of this clade contained unusual inserted sequences of â¼20 residues in the C-terminal region and were related to desaturases that all contained similar inserts. Deletion of the entirety of the insert in the flour beetle Δ12 desaturase abolished its activity but this was partially restored by the reintroduction of two histidine residues, suggesting the histidine(s) are required for activity but the full length insert is not. Five new desaturase activities were discovered: Δ9 desaturation of C12:0-C16:0 substrates; two unprecedented Δ5 enzymes acting on C18:0 and C16:0; Δ9 activity exclusively on C16:0 and a further stearate Δ9 desaturase. qPCR analysis ruled out a role in sex pheromone synthesis for the Δ5 and Δ9/C16:0 desaturases. The flour beetle genome has underpinned an examination of all transcribed desaturases in the organism and revealed a diversity of novel and unusual activities, an improved understanding of the evolutionary relationships among insect desaturases and sequence determinants of activity.