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1.
Am J Physiol Endocrinol Metab ; 318(3): E318-E329, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961704

RESUMO

Browning of white adipose tissue (WAT) has been recognized as an important strategy for the treatment of obesity, insulin resistance, and diabetes. Enoyl coenzyme A hydratase 1 (ECH1) is a widely known enzyme involved in lipid metabolism. However, whether and how ECH1 is implicated in browning of WAT remain obscure. Adeno-associated, virus-mediated genetic engineering of ECH1 in adipose tissue was used in investigations in mouse models of obesity induced by a high-fat diet (HFD) or browning induced by cold exposure. Metabolic parameters showed that ECH1 overexpression decreased weight gain and improved insulin sensitivity and lipid profile after 8 wk of an HFD. Further work revealed that these changes were associated with enhanced energy expenditure and increased appearance of brown-like adipocytes in inguinal WAT, as verified by a remarkable increase in uncoupling protein 1 and thermogenic gene expression. In vitro, ECH1 induced brown fat-related gene expression in adipocytes differentiated from primary stromal vascular fractions, whereas knockdown of ECH1 reversed this effect. Mechanistically, ECH1 regulated the thermogenic program by inhibiting mammalian target of rapamycin signaling, which may partially explain the potential mechanism for ECH1 regulating adipose browning. In summary, ECH1 may participate in the pathology of obesity by regulating browning of WAT, which probably provides us with a new therapeutic strategy for combating obesity.


Assuntos
Tecido Adiposo Marrom/enzimologia , Isomerases de Ligação Dupla Carbono-Carbono/genética , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Terapia Genética/métodos , Doenças Metabólicas/terapia , Obesidade/terapia , Tecido Adiposo Marrom/crescimento & desenvolvimento , Tecido Adiposo Branco/enzimologia , Tecido Adiposo Branco/crescimento & desenvolvimento , Animais , Temperatura Baixa , Dieta Hiperlipídica , Metabolismo Energético , Engenharia Genética , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Serina-Treonina Quinases TOR/metabolismo , Termogênese , Ganho de Peso
2.
Mol Metab ; 25: 35-49, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31060926

RESUMO

OBJECTIVE: Brown adipose tissue (BAT) is important for thermoregulation in many mammals. Uncoupling protein 1 (UCP1) is the critical regulator of thermogenesis in BAT. Here we aimed to investigate the deacetylation control of BAT and to investigate a possible functional connection between UCP1 and sirtuin 3 (SIRT3), the master mitochondrial lysine deacetylase. METHODS: We carried out physiological, molecular, and proteomic analyses of BAT from wild-type and Sirt3KO mice when BAT is activated. Mice were either cold exposed for 2 days or were injected with the ß3-adrenergic agonist, CL316,243 (1 mg/kg; i.p.). Mutagenesis studies were conducted in a cellular model to assess the impact of acetylation lysine sites on UCP1 function. Cardiac punctures were collected for proteomic analysis of blood acylcarnitines. Isolated mitochondria were used for functional analysis of OXPHOS proteins. RESULTS: Our findings showed that SIRT3 absence in mice resulted in impaired BAT lipid use, whole body thermoregulation, and respiration in BAT mitochondria, without affecting UCP1 expression. Acetylome profiling of BAT mitochondria revealed that SIRT3 regulates acetylation status of many BAT mitochondrial proteins including UCP1 and crucial upstream proteins. Mutagenesis work in cells suggested that UCP1 activity was independent of direct SIRT3-regulated lysine acetylation. However, SIRT3 impacted BAT mitochondrial proteins activities of acylcarnitine metabolism and specific electron transport chain complexes, CI and CII. CONCLUSIONS: Our data highlight that SIRT3 likely controls BAT thermogenesis indirectly by targeting pathways upstream of UCP1.


Assuntos
Tecido Adiposo Marrom/metabolismo , Sirtuína 3/metabolismo , Proteína Desacopladora 1/metabolismo , 3-Hidroxiacil-CoA Desidrogenases , Acetil-CoA C-Aciltransferase , Acetilação , Tecido Adiposo Marrom/patologia , Antagonistas de Receptores Adrenérgicos beta 3/efeitos adversos , Animais , Composição Corporal , Regulação da Temperatura Corporal , Isomerases de Ligação Dupla Carbono-Carbono , Carnitina/análogos & derivados , Carnitina/farmacologia , Enoil-CoA Hidratase , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Modelos Animais , Mutagênese , Fosforilação Oxidativa , Proteômica , Racemases e Epimerases , Sirtuína 3/genética , Termogênese/fisiologia
3.
Int J Mol Sci ; 19(12)2018 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-30567295

RESUMO

BACKGROUND: Lipid expression is increased in the atrial myocytes of mitral regurgitation (MR) patients. This study aimed to investigate key regulatory genes and mechanisms of atrial lipotoxic myopathy in MR. METHODS: The HL-1 atrial myocytes were subjected to uniaxial cyclic stretching for eight hours. Fatty acid metabolism, lipoprotein signaling, and cholesterol metabolism were analyzed by PCR assay (168 genes). RESULTS: The stretched myocytes had significantly larger cell size and higher lipid expression than non-stretched myocytes (all p < 0.001). Fatty acid metabolism, lipoprotein signaling, and cholesterol metabolism in the myocytes were analyzed by PCR assay (168 genes). In comparison with their counterparts in non-stretched myocytes, seven genes in stretched monocytes (Idi1, Olr1, Nr1h4, Fabp2, Prkag3, Slc27a5, Fabp6) revealed differential upregulation with an altered fold change >1.5. Nine genes in stretched monocytes (Apoa4, Hmgcs2, Apol8, Srebf1, Acsm4, Fabp1, Acox2, Acsl6, Gk) revealed differential downregulation with an altered fold change <0.67. Canonical pathway analysis, using Ingenuity Pathway Analysis software, revealed that the only genes in the "superpathway of cholesterol biosynthesis" were Idi1 (upregulated) and Hmgcs2 (downregulated). The fraction of stretched myocytes expressing Nile red was significantly decreased by RNA interference of Idi1 (p < 0.05) and was significantly decreased by plasmid transfection of Hmgcs2 (p = 0.004). CONCLUSIONS: The Idi1 and Hmgcs2 genes have regulatory roles in atrial lipotoxic myopathy associated with atrial enlargement.


Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/genética , Hidroximetilglutaril-CoA Sintase/genética , Metabolismo dos Lipídeos/genética , Insuficiência da Valva Mitral/genética , Linhagem Celular , Colesterol/genética , Colesterol/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica/genética , Átrios do Coração/metabolismo , Átrios do Coração/fisiopatologia , Hemiterpenos , Humanos , Lipídeos/genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Insuficiência da Valva Mitral/metabolismo , Insuficiência da Valva Mitral/fisiopatologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Transdução de Sinais
4.
Sci Rep ; 8(1): 14696, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30279540

RESUMO

Pyrophosphate (PPi) is produced by anabolic reactions and serves as an energy donor in the cytosol of plant cells; however, its accumulation to toxic levels disrupts several common biosynthetic pathways and is lethal. Before acquiring photosynthetic capacity, young seedlings must endure a short but critical heterotrophic period, during which they are nourished solely by sugar produced from seed reserves by the anabolic process of gluconeogenesis. Previously, we reported that excess PPi in H+-PPase-knockout fugu5 mutants of Arabidopsis thaliana severely compromised gluconeogenesis. However, the precise metabolic target of PPi inhibition in vivo remained elusive. Here, CE-TOF MS analyses of major metabolites characteristic of gluconeogenesis from seed lipids showed that the Glc6P;Fru6P level significantly increased and that Glc1P level was consistently somewhat higher in fugu5 compared to wild type. In contrast, the UDP-Glc level decreased significantly in the mutants. Importantly, specific removal of PPi in fugu5, and thus in AVP1pro:IPP1 transgenic lines, restored the Glc1P and the Glc6P;Fru6P levels, increased the UDP-Glc level ~2.0-fold, and subsequently increased sucrose synthesis. Given the reversible nature of the Glc1P/UDP-Glc reaction, our results indicate that UGP-Glc pyrophosphorylase is the major target when excess PPi exerts inhibitory effects in vivo. To validate our findings, we analyzed metabolite responses using a mathematical theory called structural sensitivity analysis (SSA), in which the responses of concentrations in reaction systems to perturbations in enzyme activity are determined from the structure of the network alone. A comparison of our experimental data with the results of pure structural theory predicted the existence of unknown reactions as the necessary condition for the above metabolic profiles, and confirmed the above results. Our data support the notion that H+-PPase plays a pivotal role in cytosolic PPi homeostasis in plant cells. We propose that the combination of metabolomics and SSA is powerful when seeking to identify and predict metabolic targets in living cells.


Assuntos
Arabidopsis/metabolismo , Difosfatos/metabolismo , Gluconeogênese , Uridina Difosfato Glucose/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Isomerases de Ligação Dupla Carbono-Carbono/genética , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Hemiterpenos , Pirofosfatase Inorgânica/genética , Pirofosfatase Inorgânica/metabolismo , Metabolômica , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plântula/metabolismo
5.
J Am Chem Soc ; 140(40): 12900-12908, 2018 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-30183274

RESUMO

Type 1 and type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-1 and IDI-2) catalyze the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the fundamental building blocks for biosynthesis of isoprenoid compounds. Previous studies indicate that both isoforms of IDI catalyze isomerization by a protonation-deprotonation mechanism. IDI-1 and IDI-2 are "sluggish" enzymes with turnover times of ∼10 s-1 and ∼1 s-1, respectively. We measured incorporation of deuterium into IPP and DMAPP in D2O buffer for IDI-1 and IDI-2 under conditions where newly synthesized DMAPP is immediately and irreversibly removed by coupling its release to condensation with l-tryptophan catalyzed by dimethylallyltrytophan synthase. During the course of the reactions, we detected formation of d1, d2, and d3 isotopologues of IPP and DMAPP, which were formed during up to five isomerizations between IPP and DMAPP during each turnover. The patterns for deuterium incorporation into IPP show that d2-IPP is formed in preference to d1-IPP for both enzymes. Analysis of the patterns of deuterium incorporation are consistent with a mechanism involving addition and removal of protons by a concerted asynchronous process, where addition substantially precedes removal, or a stepwise process through a short-lived (<3 ps) tertiary carbocationic intermediate. Previous work with mechanism-based inhibitors and related model studies supports a concerted asynchronous mechanism for the enzyme-catalyzed isomerizations.


Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Hemiterpenos/metabolismo , Compostos Organofosforados/metabolismo , Prótons , Streptococcus pneumoniae/enzimologia , Deutério/metabolismo , Escherichia coli/metabolismo , Isomerismo , Modelos Moleculares , Streptococcus pneumoniae/metabolismo
6.
Gene ; 679: 195-201, 2018 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-30194986

RESUMO

The aim of this study was to verify the effects of TwIDI (GenBank: KT279355.1) on triptolide and celastrol accumulation in the biosynthesis of terpenoids in Tripterygium wilfordii and the regulation of the expression of related genes in the triptolide and celastrol biosynthesis pathway. After bioinformatics analysis of TwIDI, we cloned the full-length CDS and a specific 398 bp fragment to construct overexpression and RNAi vectors, respectively. The specific amplification of hygromycin and kanamycin resistance gene fragments confirmed that the expression vectors had been successfully delivered into Tripterygium wilfordii suspension cells. qRT-PCR was used to detect the expression of TwIDI and related genes in the triptolide and celastrol biosynthesis pathway. The expression of TwIDI was increased to 157% of the control group (empty vector) in the overexpression group, and was reduced to 71% of the control group in the RNAi group. Notably, the expression of other genes in the triptolide and celastrol biosynthesis pathway also showed differences. For example, TwMCS was reduced to 62% of the control when TwIDI was overexpressed and increased to 188% in the RNAi group. The expression of TwDXS did not change significantly both during TwIDI overexpression and RNAi group. The accumulation of triptolide and celastrol in the suspension cells of Tripterygium wilfordii was detected by UPLC, revealing that the contents of triptolide and celastrol were increased 1.36- and 1.20-fold over the control group in the overexpression group, and decreased to 0.16 and 0.36 of the control group in the RNAi group. Based on these findings, the effect on the accumulation of active terpenoids in Tripterygium wilfordii and the feedback regulation of genes in the triptolide and celastrol biosynthesis pathway was verified through TwIDI overexpression and RNAi experiments.


Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/genética , Diterpenos/metabolismo , Fenantrenos/metabolismo , Tripterygium/genética , Triterpenos/metabolismo , Vias Biossintéticas , Regulação para Baixo , Compostos de Epóxi/metabolismo , Regulação da Expressão Gênica de Plantas , Hemiterpenos , Filogenia , Proteínas de Plantas/genética , Interferência de RNA , Tripterygium/metabolismo , Regulação para Cima
7.
ACS Synth Biol ; 7(9): 2308-2316, 2018 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-30145882

RESUMO

Isoprene, as a versatile bulk chemical, has wide industrial applications. Here, we attempted to improve isoprene biosynthesis in Saccharomyces cerevisiae by simultaneous strengthening of precursor supply and conversion via a combination of pathway compartmentation and protein engineering. At first, a superior isoprene synthase mutant ISPSLN was created by saturation mutagenesis, leading to almost 4-fold improvement in isoprene production. Subsequent introduction of ISPSLN to strains with strengthened precursor supply in either cytoplasm or mitochondria implied an imperfect match between the synthesis and conversion of the isopentenyl pyrophosphate (IPP)/dimethylallyl diphosphate (DMAPP) pool. To reconstruct metabolic balance between the upstream and downstream flux, additional copies of diphosphomevalonate decarboxylase gene ( MVD1) and isopentenyl-diphosphate delta-isomerase gene ( IDI1) were introduced into the cytoplasmic and mitochondrial engineered strains. Finally, the diploid strain created by mating the above haploid strains produced 11.9 g/L of isoprene, the highest ever reported in eukaryotic cells.


Assuntos
Hemiterpenos/biossíntese , Saccharomyces cerevisiae/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Butadienos/química , Isomerases de Ligação Dupla Carbono-Carbono/genética , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Carboxiliases/genética , Carboxiliases/metabolismo , Citoplasma/metabolismo , Hemiterpenos/química , Hemiterpenos/metabolismo , Mitocôndrias/metabolismo , Mutagênese , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Engenharia de Proteínas
8.
Biochem Biophys Res Commun ; 499(3): 403-409, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29526751

RESUMO

Metabolic disorders, including obesity, non-alcoholic fatty liver disease (NAFLD), metabolic syndrome and diabetes, are complex and progressive diseases. Enoyl coenzyme A hydratase 1 (Ech1) is an enzyme that participates in mitochondrial fatty acid ß-oxidation; however, little is known regarding the significance of Ech1 in the pathogenesis of metabolic disorders. Here, we report that high-fat-diet (HFD)-induced and genetic obesity could increase Ech1 expression in mouse liver. The overexpression of Ech1 using adeno-associated virus (AAV2/8) ameliorated HFD-induced liver lipid accumulation and accompanying liver injury. Additionally, Ech1 overexpression resulted in improved dyslipidemia and insulin resistance in HFD-fed mice. Further, the studies revealed that Ech1 could directly inhibit lipogenesis gene expressions and attenuate the insulin pathway induced by an HFD. Together, our results demonstrate that Ech1 protects against HFD-induced hepatic steatosis and insulin resistance and that its inhibitory effects on lipogenesis and insulin signaling may partly explain its role in metabolic disorders.


Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Resistência à Insulina , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Animais , Dieta Hiperlipídica , Dislipidemias/complicações , Dislipidemias/enzimologia , Dislipidemias/patologia , Insulina/metabolismo , Lipogênese , Fígado/lesões , Fígado/metabolismo , Fígado/patologia , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade/enzimologia , Obesidade/genética , Transdução de Sinais
9.
Mol Cell ; 69(4): 689-698.e7, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29429925

RESUMO

Endothelial-to-mesenchymal transition (EndoMT) is a cellular process often initiated by the transforming growth factor ß (TGF-ß) family of ligands. Although required for normal heart valve development, deregulated EndoMT is linked to a wide range of pathological conditions. Here, we demonstrate that endothelial fatty acid oxidation (FAO) is a critical in vitro and in vivo regulator of EndoMT. We further show that this FAO-dependent metabolic regulation of EndoMT occurs through alterations in intracellular acetyl-CoA levels. Disruption of FAO via conditional deletion of endothelial carnitine palmitoyltransferase II (Cpt2E-KO) augments the magnitude of embryonic EndoMT, resulting in thickening of cardiac valves. Consistent with the known pathological effects of EndoMT, adult Cpt2E-KO mice demonstrate increased permeability in multiple vascular beds. Taken together, these results demonstrate that endothelial FAO is required to maintain endothelial cell fate and that therapeutic manipulation of endothelial metabolism could provide the basis for treating a growing number of EndoMT-linked pathological conditions.


Assuntos
Carnitina O-Palmitoiltransferase/fisiologia , Endotélio Vascular/metabolismo , Transição Epitelial-Mesenquimal , Ácidos Graxos/química , 3-Hidroxiacil-CoA Desidrogenases , Acetilcoenzima A/metabolismo , Acetil-CoA C-Aciltransferase , Animais , Isomerases de Ligação Dupla Carbono-Carbono , Células Cultivadas , Endotélio Vascular/citologia , Enoil-CoA Hidratase , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Racemases e Epimerases , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo
10.
Angew Chem Int Ed Engl ; 57(7): 1879-1882, 2018 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-29232490

RESUMO

We show that CO2 can be converted by an engineered "Knallgas" bacterium (Cupriavidus necator) into the terpene α-humulene. Heterologous expression of the mevalonate pathway and α-humulene synthase resulted in the production of approximately 10 mg α-humulene per gram cell dry mass (CDW) under heterotrophic conditions. This first example of chemolithoautotrophic production of a terpene from carbon dioxide, hydrogen, and oxygen is a promising starting point for the production of different high-value terpene compounds from abundant and simple raw materials. Furthermore, the production system was used to produce 17 mg α-humulene per gram CDW from CO2 and electrical energy in microbial electrosynthesis (MES) mode. Given that the system can convert CO2 by using electrical energy from solar energy, it opens a new route to artificial photosynthetic systems.


Assuntos
Dióxido de Carbono/metabolismo , Cupriavidus necator/química , Sesquiterpenos/metabolismo , Terpenos/metabolismo , Processos Autotróficos , Dióxido de Carbono/química , Isomerases de Ligação Dupla Carbono-Carbono/genética , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Cupriavidus necator/genética , Cupriavidus necator/metabolismo , Hemiterpenos , Hidrogênio/química , Hidrogênio/metabolismo , Isomerases/genética , Isomerases/metabolismo , Engenharia Metabólica , Sesquiterpenos Monocíclicos , Oxigênio/química , Oxigênio/metabolismo , Proteínas de Plantas/genética , Sesquiterpenos/química , Energia Solar , Terpenos/química
11.
Curr Genet ; 64(2): 417-422, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29043484

RESUMO

The precise and controlled regulation of gene expression at transcriptional and post-transcriptional levels is crucial for the eukaryotic cell survival and functions. In eukaryotes, more than 100 types of post-transcriptional RNA modifications have been identified. The N6-methyladenosine (m6A) modification in mRNA is among the most common post-transcriptional RNA modifications known in eukaryotic organisms, and the m6A RNA modification can regulate gene expression. The role of yeast m6A methyltransferase (Ime4) in meiosis, sporulation, triacylglycerol metabolism, vacuolar morphology, and mitochondrial functions has been reported. Stress triggers triacylglycerol accumulation as lipid droplets. Lipid droplets are physically connected to the different organelles such as endoplasmic reticulum, mitochondria, and peroxisomes. However, the physiological relevance of these physical interactions remains poorly understood. In yeast, peroxisome is the sole site of fatty acid ß-oxidation. The metabolic status of the cell readily governs the number and physiological function of peroxisomes. Under low-glucose or stationary-phase conditions, peroxisome biogenesis and proliferation increase in the cells. Therefore, we hypothesized a possible role of Ime4 in the peroxisomal functions. There is no report on the role of Ime4 in peroxisomal biology. Here, we report that IME4 gene deletion causes peroxisomal dysfunction under stationary-phase conditions in Saccharomyces cerevisiae; besides, the ime4Δ cells showed a significant decrease in the expression of the key genes involved in peroxisomal ß-oxidation compared to the wild-type cells. Therefore, identification and determination of the target genes of Ime4 that are directly involved in the peroxisomal biogenesis, morphology, and functions will pave the way to better understand the role of m6A methylation in peroxisomal biology.


Assuntos
Adenosina/análogos & derivados , Ácidos Graxos/genética , Metiltransferases/genética , Peroxissomos/genética , Proteínas de Saccharomyces cerevisiae/genética , 3-Hidroxiacil-CoA Desidrogenases/genética , Acetil-CoA C-Aciltransferase/genética , Adenosina/genética , Adenosina/metabolismo , Isomerases de Ligação Dupla Carbono-Carbono/genética , Enoil-CoA Hidratase/genética , Ácidos Graxos/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Metabolismo dos Lipídeos/genética , Metiltransferases/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Peroxissomos/enzimologia , Processamento Pós-Transcricional do RNA/genética , Racemases e Epimerases/genética , Saccharomyces cerevisiae/genética , Vacúolos/enzimologia , Vacúolos/genética
12.
Cancer Lett ; 409: 104-115, 2017 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-28923398

RESUMO

The class III deacetylase sirtuin 1 (SIRT1), a member of the sirtuin family proteins, plays a key role in many types of cancers including colorectal cancer (CRC). Here we report that SIRT1 suppressed CRC metastasis in vitro and in vivo as a negative regulator for miR-15b-5p transcription. Mechanistically, SIRT1 impaired regulatory effects of activator protein (AP-1) on miR-15b-5p trans-activation through deacetylation of AP-1. Importantly, acyl-CoA oxidase 1 (ACOX1), a key enzyme of the fatty acid oxidation (FAO) pathway, was found as a direct target for miR-15b-5p. SIRT1 expression was positively correlated with ACOX1 expression in CRC cells and in xenografts. Moreover, ACOX1 overexpression attenuated the augmentation of migration and invasion of CRC cells by miR-15b-5p overexpression. In conclusion, our study demonstrated a functional role of the SIRT1/miR-15b-5p/ACOX1 axis in CRC metastasis and suggested a potential target for metastatic CRC therapy.


Assuntos
Neoplasias Colorretais/genética , MicroRNAs/genética , Sirtuína 1/genética , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Acetil-CoA C-Aciltransferase/metabolismo , Animais , Células CACO-2 , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Enoil-CoA Hidratase/metabolismo , Células HCT116 , Células HT29 , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/metabolismo , Metástase Neoplásica , Racemases e Epimerases/metabolismo , Transdução de Sinais , Sirtuína 1/metabolismo , Transcrição Genética , Transfecção
13.
J Gen Appl Microbiol ; 63(5): 287-295, 2017 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-28954964

RESUMO

To achieve an efficient production of geraniol and its derivatives in Escherichia coli, we aimed to improve the activity of geraniol synthase (GES) through a single round of mutagenesis and screening for higher substrate consumption. We isolated GES variants that outperform their parent in geraniol production. The analysis of GES variants indicated that the expression level of GES was the bottleneck for geraniol synthesis. Over-expression of the mutant GESM53 with a 5'-untranslated sequence designed for high translational efficiency, along with the additional expression of mevalonate pathway enzymes, isopentenyl pyrophosphate isomerase, and geranyl pyrophosphate synthase, yielded 300 mg/L/12 h geraniol and its derivatives (>1000 mg/L/42 h in total) in a shaking flask.


Assuntos
Evolução Molecular Direcionada , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Escherichia coli/genética , Geraniltranstransferase , Terpenos/metabolismo , Monoterpenos Acíclicos , Isomerases de Ligação Dupla Carbono-Carbono/genética , Carotenoides/metabolismo , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Geraniltranstransferase/biossíntese , Geraniltranstransferase/genética , Hemiterpenos , Ensaios de Triagem em Larga Escala , Engenharia Metabólica , Mutagênese , Iniciação Traducional da Cadeia Peptídica/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Fosfatos de Poli-Isoprenil , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Especificidade por Substrato
14.
Arch Biochem Biophys ; 632: 47-58, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28577910

RESUMO

The chemical versatility of the flavin coenzyme is nearly unparalleled in enzyme catalysis. An interesting illustration of this versatility can be found in the reaction catalyzed by the type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) - an enzyme that interconverts the two essential isoprene units (isopentenyl pyrophosphate and dimethylallyl pyrophosphate) that are needed to initiate the biosynthesis of all isoprenoids. Over the past decade, a variety of biochemical, spectroscopic, structural and mechanistic studies of IDI-2 have provided mounting evidence that the flavin coenzyme of IDI-2 acts in a most unusual manner - as an acid/base catalyst to mediate a 1,3-proton addition/elimination reaction. While not entirely without precedent, IDI-2 is by far the most extensively studied flavoenzyme that employs flavin-mediated acid/base catalysis. Thus, IDI-2 serves as an important mechanistic model for understanding this often overlooked, but potentially widespread reactivity of flavin coenzymes. This review details the most pertinent studies that have contributed to the development of mechanistic proposals for this highly unusual flavoenzyme, and discusses future experiments that may be able to clarify remaining uncertainties in the chemical mechanism of IDI-2.


Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/química , Flavoproteínas/química , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Flavoproteínas/metabolismo , Hemiterpenos/química , Hemiterpenos/metabolismo , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , Terpenos/química , Terpenos/metabolismo
15.
Mol Nutr Food Res ; 61(10)2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28608394

RESUMO

SCOPE: The aim of this work was to study the urinary metabolomics changes of participants that consumed beer, nonalcoholic beer (na-beer), and gin. METHODS AND RESULTS: Thirty-three males at high cardiovascular risk between 55 and 75 years old participated in an open, randomized, crossover, controlled trial with three nutritional interventions consisting of beer, na-beer, and gin for 4 wk. Diet and physical activity was monitored throughout the study and compliance was assessed by measurement of urinary isoxanthohumol. Metabolomic analysis was performed in urine samples by LC coupled to an LTQ-Orbitrap mass spectrometer combined with univariate and multivariate statistical analysis. Ten metabolites were identified. Eight were exogenous metabolites related to beer, na-beer, or gin consumption, but two of them were related to endogenic changes: hydroxyadipic acid linked to fatty acid oxidation, and 4-guanidinobutanoic acid, which correlated with a decrease in urinary creatinine. Plasmatic acylcarnitines were quantified by targeted MS. A regular and moderate consumption of beer and na-beer decreased stearoylcarnitine concentrations. CONCLUSION: Humulinone and 2,3-dihydroxy-3-methylvaleric acid showed to be potential biomarkers of beer and na-beer consumption. Moreover, the results of this trial provide new evidence that the nonalcoholic fraction of beer may increase fatty oxidation.


Assuntos
Cerveja/efeitos adversos , Biomarcadores/urina , Doenças Cardiovasculares/urina , Metaboloma , Metabolômica , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Acetil-CoA C-Aciltransferase/metabolismo , Adipatos/sangue , Idoso , Consumo de Bebidas Alcoólicas , Bebidas , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Carnitina/análogos & derivados , Carnitina/sangue , Creatinina/urina , Estudos Cross-Over , Dieta , Enoil-CoA Hidratase/metabolismo , Exercício Físico , Humanos , Masculino , Pessoa de Meia-Idade , Cooperação do Paciente , Ácidos Pentanoicos/urina , Racemases e Epimerases/metabolismo , Fatores de Risco , Xantonas/urina
16.
Arch Pediatr ; 24(8): 777-782, 2017 Aug.
Artigo em Francês | MEDLINE | ID: mdl-28647472

RESUMO

Acute fatty liver of pregnancy (AFLP) is a rare liver disease unique to pregnancy that can lead to acute liver failure. The prognosis, initially often fatal for both mother and child, has been improved by prompt delivery. The diagnosis should be highly suspected if the mother presents epigastric pain, nausea and/or vomiting, or polyuria-polydipsia in the third trimester of pregnancy. AFLP has been found associated with a genetic deficiency of fatty acid beta-oxidation, which may cause sudden death in infancy. Consequently, the mother and her newborn should undergo screening for this deficiency.


Assuntos
Parto Obstétrico , Ácidos Graxos/metabolismo , Fígado Gorduroso/diagnóstico , Fígado Gorduroso/metabolismo , Mitocôndrias/metabolismo , Complicações na Gravidez/diagnóstico , Complicações na Gravidez/metabolismo , Terceiro Trimestre da Gravidez , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Acetil-CoA C-Aciltransferase/metabolismo , Adulto , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Enoil-CoA Hidratase/metabolismo , Medicina Baseada em Evidências , Fígado Gorduroso/genética , Fígado Gorduroso/terapia , Feminino , Humanos , Recém-Nascido , Gravidez , Complicações na Gravidez/genética , Complicações na Gravidez/terapia , Resultado da Gravidez , Prognóstico , Racemases e Epimerases/metabolismo , Fatores de Risco
17.
Plant J ; 91(3): 466-479, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28440881

RESUMO

We recently characterized a gene-terpene network that is associated with artemisinin biosynthesis in self-pollinated (SP) Artemisia annua, an effective antimalarial plant. We hypothesize that an alteration of gene expression in the network may improve the production of artemisinin and its precursors. In this study, we cloned an isopentenyl pyrophosphate isomerase (IPPI) cDNA, AaIPPI1, from Artemisia annua (Aa). The full-length cDNA encodes a type-I IPPI containing a plastid transit peptide (PTP) at its amino terminus. After the removal of the PTP, the recombinant truncated AaIPPI1 isomerized isopentenyl pyrophosphate (IPP) to dimethyl allyl pyrophosphate (DMAPP) and vice versa. The steady-state equilibrium ratio of IPP/DMAPP in the enzymatic reactions was approximately 1:7. The truncated AaIPPI1 was overexpressed in the cytosol of the SP A. annua variety. The leaves of transgenic plants produced approximately 4% arteannuin B (g g-1 , dry weight, dw) and 0.17-0.25% artemisinin (g g-1 , dw), the levels of which were significantly higher than those in the leaves of wild-type plants. In addition, transgenic plants showed an increase in artemisinic acid production of more than 1% (g g-1 , dw). In contrast, isoprene formation was significantly reduced in transgenic plants. These results provide evidence that overexpression of AaIPPI1 in the cytosol can lead to metabolic alterations of terpenoid biosynthesis, and show that these transgenic plants have the potential to yield high production levels of arteannuin B as a new precursor source for artemisinin.


Assuntos
Artemisia annua/enzimologia , Artemisia annua/metabolismo , Artemisininas/metabolismo , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Citosol/enzimologia , Citosol/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/metabolismo , Artemisia annua/genética , Isomerases de Ligação Dupla Carbono-Carbono/genética , Hemiterpenos , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética
18.
Biotechnol J ; 12(4)2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28217906

RESUMO

Taxol (paclitaxel) is a diterpenoid compound with significant and extensive applications in the treatment of cancer. The production of Taxol and relevant intermediates by engineered microbes is an attractive alternative to the semichemical synthesis of Taxol. In this study, based on a previously developed platform, the authors first established taxadiene production in mutant E. coli T2 and T4 by engineering of the mevalonate (MVA) pathway. The authors then developed an Agrobacterium tumefaciens-mediated transformation (ATMT) method and verified the strength of heterologous promoters in Alternaria alternata TPF6. The authors next transformed the taxadiene-producing platform into A. alternata TPF6, and the MVA pathway was engineered, with introduction of the plant taxadiene-forming gene. Notably, by co-overexpression of isopentenyl diphosphate isomerase (Idi), a truncated version of 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1), and taxadiene synthase (TS), the authors could detect 61.9 ± 6.3 µg/L taxadiene in the engineered strain GB127. This is the first demonstration of taxadiene production in filamentous fungi, and the approach presented in this study provides a new method for microbial production of Taxol. The well-established ATMT method and the known promoter strengths facilitated further engineering of taxaenes in this fungus.


Assuntos
Alcenos/metabolismo , Diterpenos/metabolismo , Engenharia Metabólica , Ácido Mevalônico/metabolismo , Neoplasias/tratamento farmacológico , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Alcenos/uso terapêutico , Alternaria/genética , Alternaria/metabolismo , Isomerases de Ligação Dupla Carbono-Carbono/biossíntese , Diterpenos/uso terapêutico , Endófitos/genética , Endófitos/metabolismo , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Proteína HMGB1/biossíntese , Hemiterpenos , Humanos , Isomerases/biossíntese , Transformação Bacteriana/genética
19.
J Lipid Res ; 58(1): 15-30, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27836993

RESUMO

Brown adipose tissue uptake of glucose and fatty acids is very high during nonshivering thermogenesis. Adrenergic stimulation markedly increases glucose uptake, de novo lipogenesis, and FA oxidation simultaneously. The mechanism that enables this concerted response has hitherto been unknown. Here, we find that in primary brown adipocytes and brown adipocyte-derived cell line (IMBAT-1), acute inhibition and longer-term knockdown of DGAT2 links the increased de novo synthesis of fatty acids from glucose to a pool of TAG that is simultaneously hydrolyzed, providing FA for mitochondrial oxidation. DGAT1 does not contribute to this pathway, but uses exogenous FA and glycerol to synthesize a functionally distinct pool of TAG to which DGAT2 also contributes. The DGAT2-dependent channelling of 14C from glucose into TAG and CO2 was reproduced in ß3-agonist-stimulated primary brown adipocytes. Knockdown of DGAT2 in IMBAT-1 affected the mRNA levels of UCP1 and genes important in FA activation and esterification. Therefore, in ß3-agonist activated brown adipocytes, DGAT2 specifically enables channelling of de novo synthesized FA into a rapidly mobilized pool of TAG, which is simultaneously hydrolyzed to provide substrates for mitochondrial fatty acid oxidation.


Assuntos
Adipócitos Marrons/metabolismo , Diacilglicerol O-Aciltransferase/genética , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos/genética , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Acetil-CoA C-Aciltransferase/metabolismo , Animais , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Linhagem Celular , Enoil-CoA Hidratase/metabolismo , Esterificação , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Lipogênese/genética , Camundongos , Oxirredução , Racemases e Epimerases/metabolismo , Triglicerídeos/metabolismo , Proteína Desacopladora 1/genética
20.
Biochemistry ; 55(30): 4229-38, 2016 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-27379573

RESUMO

Type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the isoprenoid biosynthetic pathway. The enzyme from Streptomyces pneumoniae (spIDI-2) is a homotetramer in solution with behavior, including a substantial increase in the rate of FMN reduction by NADPH in the presence of IPP, suggesting that substrate binding at one subunit alters the kinetic and binding properties of another. We now report the construction of catalytically active monomeric spIDI-2. The monomeric enzyme contains a single-point mutation (N37A) and a six-residue C-terminal deletion that preserves the secondary structure of the subunits in the wild-type (wt) homotetramer. UV-vis spectra of the enzyme-bound flavin mononucleotide (FMN) cofactor in FMNox, FMNred, and FMNred·IPP/DMAPP states are the same for monomeric and wt homotetrameric spIDI-2. The mutations in monomeric IDI-2 lower the melting temperature of the protein by 20 °C and reduce the binding affinities of FMN and IDI by 40-fold but have a minimal effect on kcat. Stopped-flow kinetic studies of monomeric spIDI-2 showed that the rate of reduction of FMN by NADH (k = 1.64 × 10(-3) s(-1)) is substantially faster when IPP is added to the monomeric enzyme (k = 0.57 s(-1)), similar to behavior seen for wt-spIDI-2. Our results indicate that cooperative interactions among subunits in the wt homotetramer are not responsible for the increased rate of reduction of spIDI-2·FMN by NADH, and two possible scenarios for the enhancement are suggested.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Isomerases de Ligação Dupla Carbono-Carbono/química , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Proteínas de Bactérias/genética , Isomerases de Ligação Dupla Carbono-Carbono/genética , Hemiterpenos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Engenharia de Proteínas , Estrutura Quaternária de Proteína , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/genética , Streptomyces/enzimologia , Streptomyces/genética
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