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1.
Nat Commun ; 11(1): 4891, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994417

RESUMO

Peripheral sensory neurons regenerate their axon after nerve injury to enable functional recovery. Intrinsic mechanisms operating in sensory neurons are known to regulate nerve repair, but whether satellite glial cells (SGC), which completely envelop the neuronal soma, contribute to nerve regeneration remains unexplored. Using a single cell RNAseq approach, we reveal that SGC are distinct from Schwann cells and share similarities with astrocytes. Nerve injury elicits changes in the expression of genes related to fatty acid synthesis and peroxisome proliferator-activated receptor (PPARα) signaling. Conditional deletion of fatty acid synthase (Fasn) in SGC impairs axon regeneration. The PPARα agonist fenofibrate rescues the impaired axon regeneration in mice lacking Fasn in SGC. These results indicate that PPARα activity downstream of FASN in SGC contributes to promote axon regeneration in adult peripheral nerves and highlight that the sensory neuron and its surrounding glial coat form a functional unit that orchestrates nerve repair.


Assuntos
Regeneração Nervosa , Neuroglia/citologia , Células Receptoras Sensoriais/citologia , Animais , Axônios/fisiologia , Proliferação de Células , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo , Traumatismos dos Nervos Periféricos/genética , Traumatismos dos Nervos Periféricos/metabolismo , Traumatismos dos Nervos Periféricos/fisiopatologia , Nervos Periféricos/crescimento & desenvolvimento , Nervos Periféricos/metabolismo , Nervos Periféricos/fisiopatologia , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais
2.
Sci Rep ; 10(1): 14733, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32895447

RESUMO

Nitazoxanide (NTZ) is effective against helminths and numerous microorganisms, including bacteria and viruses. In vivo, NTZ is metabolized into Tizoxanide (TIZ), which is the active circulating metabolite. With the emergence of SARS-Cov-2 as a Pandemic agent, NTZ became one of the molecules already approved for human use to engage clinical trials, due to results in vitro showing that NTZ was highly effective against the SARS-Cov-2, agent of COVID-19. There are currently several ongoing clinical trials mainly in the USA and Brazil involving NTZ due not only to the in vitro results, but also for its long-known safety. Here, we study the response of Vero cells to TIZ treatment and unveil possible mechanisms for its antimicrobial effect, using a label-free proteomic approach (LC/MS/MS) analysis to compare the proteomic profile between untreated- and TIZ-treated cells. Fifteen differentially expressed proteins were observed related to various biological processes, including translation, intracellular trafficking, RNA processing and modification, and signal transduction. The broad antimicrobial range of TIZ points towards its overall effect in lowering cell metabolism and RNA processing and modification. The decreased levels of FASN, HNRNPH and HNRNPK with the treatment appear to be important for antiviral activity.


Assuntos
Anti-Infecciosos/farmacologia , Proteoma/efeitos dos fármacos , Tiazóis/farmacologia , Animais , Chlorocebus aethiops , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Proteoma/genética , Proteoma/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Células Vero
3.
J Food Sci ; 85(6): 1915-1923, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32460375

RESUMO

Polar components (PCs) are produced during the frying of oil, affecting the quality of edible oil and posing a hazard to human health. In this study, C57 mice were fed a high-fat (HF) diet containing purified PCs for nine weeks. Their effects on lipid metabolism and liver function in animals were analyzed. Our results indicated that the contents of total PCs and saturated fatty acid increased from 6.07 ± 0.6% and 58.27 ± 0.35% to 19.17 ± 1.8% and 69.91 ± 0.51%, respectively (P < 0.01). PC intake resulted an 18.56% higher liver index in mice than that in the HF group. The PC group had the highest malondialdehyde (MDA) content (1.94 ± 0.11 nmol/mg protein) and the liver nonalcoholic fatty liver disease (NAFLD) activity score (NAS) was 4, which already showed NAFLD characteristics. In addition, the expression levels of lipid metabolism-related genes, including sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthetase (FAS), peroxisome proliferator-activated receptor-alpha, and peroxisome acyl-CoA oxidase 1, indicated that PC increased hepatic lipid accumulation by upregulating the transcriptional level of fat synthesis genes and further leads to liver damage by affecting mitochondrial function. Our results provided important information about the effects of PCs produced in the frying process of PO on animal health, which is critical for assessing the biosafety of fried products. PRACTICAL APPLICATION: The research will help promote the industrial upgrading of fried foods and help consumers build healthy lifestyles.


Assuntos
Metabolismo dos Lipídeos , Fígado/metabolismo , Óleo de Palmeira/química , Óleo de Palmeira/metabolismo , Animais , Culinária , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/metabolismo , Temperatura Alta , Humanos , Masculino , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo , Óleo de Palmeira/efeitos adversos , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
4.
Nat Chem Biol ; 16(7): 776-782, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32367018

RESUMO

In type II polyketide synthases (PKSs), the ketosynthase-chain length factor (KS-CLF) complex catalyzes polyketide chain elongation with the acyl carrier protein (ACP). Highly reducing type II PKSs, represented by IgaPKS, produce polyene structures instead of the well-known aromatic skeletons. Here, we report the crystal structures of the Iga11-Iga12 (KS-CLF) heterodimer and the covalently cross-linked Iga10=Iga11-Iga12 (ACP=KS-CLF) tripartite complex. The latter structure revealed the molecular basis of the interaction between Iga10 and Iga11-Iga12, which differs from that between the ACP and KS of Escherichia coli fatty acid synthase. Furthermore, the reaction pocket structure and site-directed mutagenesis revealed that the negative charge of Asp 113 of Iga11 prevents further condensation using a ß-ketoacyl product as a substrate, which distinguishes IgaPKS from typical type II PKSs. This work will facilitate the future rational design of PKSs.


Assuntos
Proteína de Transporte de Acila/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Ácido Graxo Sintases/química , Policetídeo Sintases/química , Policetídeos/química , Proteína de Transporte de Acila/genética , Proteína de Transporte de Acila/metabolismo , Biocatálise , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/enzimologia , Streptomyces/genética , Especificidade por Substrato
5.
Gene ; 741: 144516, 2020 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-32119914

RESUMO

To study the influence of the PGC-1ß gene on chicken adipocyte proliferation and differentiation, we constructed RNA interference (RNAi) vectors that target the PGC-1ß gene and transfected these vectors into adipocytes. Oil Red O staining and a CCK-8 cell kit were used to determine cell triglyceride accumulation status and cell proliferation after transfection, respectively. The mRNA abundances of PGC-1ß and adipocyte-differentiation-related genes (PPARγ, C/EBPα, SREBP-1c, FAS, and A-FABP) were detected by real-time PCR. The results showed that the mRNA and protein abundances of PGC-1ß in PGC-1ß-shRNA transfected adipocytes were significantly lower than those in the control. Interference decreased cell differentiation, but did not depress the cell proliferation. PGC-1ß interference impeded the triglyceride accumulation, the mRNA expression levels of nuclear receptors PPARγ and SREBP-1c, and fatty acid synthetase (FAS), and both proteins PPARγ and SREBP-1c, and the fatty acids transporting protein A-FABP. Generally, PGC-1ß modulated the cell differentiation and triglyceride accumulation in chicken adipocytes.


Assuntos
Adipócitos/metabolismo , Adipogenia/genética , Diferenciação Celular/genética , Receptores Ativados por Proliferador de Peroxissomo/genética , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proliferação de Células/genética , Galinhas/genética , Galinhas/crescimento & desenvolvimento , Ácido Graxo Sintases/genética , Proteínas de Ligação a Ácido Graxo/genética , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , PPAR gama/genética , RNA Mensageiro , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Triglicerídeos/metabolismo , Receptor fas/genética
6.
Biochem J ; 477(2): 491-508, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31922183

RESUMO

Acyl carrier proteins (ACPs) are small helical proteins found in all kingdoms of life, primarily involved in fatty acid and polyketide biosynthesis. In eukaryotes, ACPs are part of the fatty acid synthase (FAS) complex, where they act as flexible tethers for the growing lipid chain, enabling access to the distinct active sites in FAS. In the type II synthesis systems found in bacteria and plastids, these proteins exist as monomers and perform various processes, from being a donor for synthesis of various products such as endotoxins, to supplying acyl chains for lipid A and lipoic acid FAS (quorum sensing), but also as signaling molecules, in bioluminescence and activation of toxins. The essential and diverse nature of their functions makes ACP an attractive target for antimicrobial drug discovery. Here, we report the structure, dynamics and evolution of ACPs from three human pathogens: Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii, which could facilitate the discovery of new inhibitors of ACP function in pathogenic bacteria.


Assuntos
Proteína de Transporte de Acila/ultraestrutura , Infecções Bacterianas/microbiologia , Ácido Graxo Sintases/ultraestrutura , Conformação Proteica , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/genética , Sequência de Aminoácidos/genética , Infecções Bacterianas/tratamento farmacológico , Borrelia burgdorferi/química , Borrelia burgdorferi/patogenicidade , Borrelia burgdorferi/ultraestrutura , Brucella melitensis/química , Brucella melitensis/patogenicidade , Brucella melitensis/ultraestrutura , Domínio Catalítico , Ácido Graxo Sintases/química , Ácido Graxo Sintases/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Lipídeo A/química , Lipídeo A/genética , Simulação de Dinâmica Molecular , Complexos Multienzimáticos , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica/genética , Percepção de Quorum/genética , Rickettsia prowazekii/química , Rickettsia prowazekii/patogenicidade , Rickettsia prowazekii/ultraestrutura
7.
PLoS One ; 15(1): e0227666, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31945099

RESUMO

Species-specific sex pheromones play key roles in moth sexual communication. Although the general pathway of Type-I sex pheromone biosynthesis is well established, only a handful of genes encoding enzymes involved in this pathway have been characterized. Streltzoviella insularis is a destructive wood-boring pest of many street trees in China, and the female sex pheromone of this species comprises a blend of (Z)-3-tetradecenyl acetate, (E)-3-tetradecenyl acetate, and (Z)-5-dodecenyl acetate. This organism therefore provides an excellent model for research on the diversity of genes and molecular mechanisms involved in pheromone production. Herein, we assembled the pheromone gland transcriptome of S. insularis by next-generation sequencing and identified 74 genes encoding candidate key enzymes involved in the fatty acid biosynthesis, ß-oxidation, and functional group modification. In addition, tissue expression patterns further showed that an acetyl-CoA carboxylase and two desaturases were highly expressed in the pheromone glands compared with the other tissues, indicating possible roles in S. insularis sex pheromone biosynthesis. Finally, we proposed putative S. insularis biosynthetic pathways for sex pheromone components and highlighted candidate genes. Our findings lay a solid foundation for understanding the molecular mechanisms underpinning S. insularis sex pheromone biosynthesis, and provide potential targets for disrupting chemical communication that could assist the development of novel pest control methods.


Assuntos
Genes de Insetos , Mariposas/genética , Mariposas/metabolismo , Atrativos Sexuais/biossíntese , Atrativos Sexuais/genética , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Animais , Vias Biossintéticas/genética , China , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Filogenia , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Glândulas Odoríferas/metabolismo , Análise de Sequência de RNA , Transcriptoma
8.
Am J Chin Med ; 47(8): 1885-1899, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31838869

RESUMO

Diet polyphenol can reportedly prevent the formation of breast-cancer cells. Nelumbo nucifera leaf extract (NLE) is enriched with polyphenols and has several cellular functions, such as anti-atherosclerosis, anti-inflammation, and antitumor. In this study, we investigated the role of NLE in the prevention of N-methyl-N-nitrosourea (NMU)-induced mammary tumor formation. Cotreatment with NLE significantly reduced the NMU-induced tumor incidence, number, and volume. NLE administration significantly repressed the tumor growth and weight of nude mice upon inoculation with BT-474 cancer cells. Immunohistochemical staining indicated that fatty acid synthetase, estrogen receptor (ER)-α, and phosphorylated ER-α were obviously reduced in the cancer part of BT-474 inoculated nude mice upon administration of 2% NLE. Western blot analysis revealed that NLE and NLPE (polyphenol-rich NLE) repressed ER-α expression and phosphorylation and decreased the phosphorylation of Her-2 without affecting their expression. Overall, NLE and NLPE exhibited more effective antitumor abilities in NMU-induced mammary cancer formation than with tamoxifen and Herceptin.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Medicamentos de Ervas Chinesas/administração & dosagem , Receptor alfa de Estrogênio/metabolismo , Ácido Graxo Sintases/genética , Nelumbo/química , Receptor ErbB-2/genética , Animais , Neoplasias da Mama/induzido quimicamente , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Regulação para Baixo/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Ácido Graxo Sintases/metabolismo , Feminino , Humanos , Metilnitrosoureia/efeitos adversos , Camundongos , Camundongos Nus , Ratos , Ratos Sprague-Dawley , Receptor ErbB-2/metabolismo
9.
Sci Rep ; 9(1): 19470, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31857635

RESUMO

Thraustochytrids of the genera Schizochytrium and Aurantiochytrium accumulate oils rich in the essential, marine n3 fatty acid docosahexaenoic acid (DHA). DHA production in Aurantiochytrium sp T66 was studied with the aim to provide more knowledge about factors that affect the DHA-productivities and the contributions of the two enzyme systems used for fatty acid synthesis in thraustochytrids, fatty acid synthetase (FAS) and PUFA-synthase. Fermentations with nitrogen starvation, which is well-known to initiate lipid accumulation in oleaginous organisms, were compared to fermentations with nitrogen in excess, obtained by oxygen limitation. The specific productivities of fatty acids originating from FAS were considerably higher under nitrogen starvation than with nitrogen in excess, while the specific productivities of DHA were the same at both conditions. Global transcriptome analysis showed significant up-regulation of FAS under N-deficient conditions, while the PUFA-synthase genes were only marginally upregulated. Neither of them was upregulated under O2-limitation where nitrogen was in excess, suggesting that N-starvation mainly affects the FAS and may be less important for the PUFA-synthase. The transcriptome analysis also revealed responses likely to be related to the generation of reducing power (NADPH) for fatty acid synthesis.


Assuntos
Ácidos Docosa-Hexaenoicos/biossíntese , Ácido Graxo Sintases/metabolismo , Estramenópilas/metabolismo , Ácido Graxo Sintases/genética , Fermentação , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Cinética , NADP/biossíntese , Nitrogênio/metabolismo , Oxigênio/metabolismo , Regulação para Cima
10.
Br J Nutr ; 122(11): 1201-1211, 2019 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-31782376

RESUMO

Disturbances in lipid metabolism are at the core of several health issues facing modern society, including fatty liver and obesity. The sterol regulatory element-binding protein 1 (SREBP-1) is one important transcription factor regulating lipid metabolism, but the relevant mechanism still remains unknown. The present study determined the transcriptional regulation of SREBP-1 and its target genes (including acetyl-CoA carboxylase α (accα), fatty acid synthase (fas) and stearoyl-CoA desaturase 1 (scd1)) in a freshwater teleost, grass carp Ctenopharyngodon idella. We cloned and characterised the 1988 bp, 2043 bp, 1632 bp and 1889 bp sequences of srebp-1, accα, scd1 and fas promoters, respectively. A cluster of putative binding sites of transcription factors, such as specific protein, yin yang 1, nuclear factor Y, sterol response elements (SRE) and enhancer box (E-box) element, were predicted on their promoter regions. Overexpression of nSREBP-1 reduced srebp-1 promoter activity, increased scd1 and fas promoter activity but did not influence accα promoter activity. The site-mutation and electrophoretic mobility shift assay analysis indicated that srebp-1, fas and scd1 promoters, but not accα promoter, possessed SRE. In Ctenopharyngodon idella kidney (CIK) cells of grass carp, nSREBP-1 overexpression significantly reduced srebp-1 mRNA expression and up-regulated miR-29 mRNA expression. The 3'UTR of srebp-1 possessed the potential miR-29 binding site and miR-29 up-regulated the luciferase activity of srebp-1 3'UTR and srebp-1 mRNA expression, implying a self-activating loop of SREBP-1 and miR-29 in grass carp. Based on the above-mentioned results, we found two novel transcriptional mechanisms for SREBP-1 in grass carp: (1) the auto-regulation sited on the SREBP-1 promoter regions was suppressive and (2) there was a self-activating loop of SREBP-1 and miR-29.


Assuntos
Carpas/metabolismo , Lipogênese/fisiologia , Proteína de Ligação a Elemento Regulador de Esterol 1/fisiologia , Acetil-CoA Carboxilase/genética , Animais , Carpas/genética , Células Cultivadas , Clonagem Molecular , Ácido Graxo Sintases/genética , Regulação da Expressão Gênica , Células Hep G2 , Humanos , Rim/química , Rim/metabolismo , Lipogênese/genética , MicroRNAs/genética , MicroRNAs/fisiologia , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA/veterinária , Estearoil-CoA Dessaturase/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Transcrição Genética/fisiologia , Transfecção
11.
Nat Commun ; 10(1): 5011, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676791

RESUMO

Upregulation of fatty acid synthase (FASN) is a common event in cancer, although its mechanistic and potential therapeutic roles are not completely understood. In this study, we establish a key role of FASN during transformation. FASN is required for eliciting the anaplerotic shift of the Krebs cycle observed in cancer cells. However, its main role is to consume acetyl-CoA, which unlocks isocitrate dehydrogenase (IDH)-dependent reductive carboxylation, producing the reductive power necessary to quench reactive oxygen species (ROS) originated during the switch from two-dimensional (2D) to three-dimensional (3D) growth (a necessary hallmark of cancer). Upregulation of FASN elicits the 2D-to-3D switch; however, FASN's synthetic product palmitate is dispensable for this process since cells satisfy their fatty acid requirements from the media. In vivo, genetic deletion or pharmacologic inhibition of FASN before oncogenic activation prevents tumor development and invasive growth. These results render FASN as a potential target for cancer prevention studies.


Assuntos
Células-Tronco Embrionárias/metabolismo , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/metabolismo , Fibroblastos/metabolismo , Neoplasias Experimentais/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Ácido Graxo Sintases/química , Ácido Graxo Sintases/genética , Feminino , Fibroblastos/citologia , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Camundongos Transgênicos , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Carga Tumoral/genética
12.
J Agric Food Chem ; 67(45): 12419-12427, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31610126

RESUMO

The liver X receptors (LXRs) are major regulators of lipogenesis, and their reduced activation by an inhibitor could be a treatment strategy for fatty liver disease. Small molecules originating from dietary food are considered suitable and attractive drug candidates for humans in terms of safety. In this study, an edible plant, Lysimachia vulgaris (LV), used as a traditional and medicinal food in East Asia was evaluated for lipogenesis decreasing effects. Activity-guided fractionation was performed, and the isolated compounds were identified using spectroscopic methods. We conducted in vitro real-time polymerase chain reaction (PCR) and Western blotting as well as histological and biochemical analyses following in vivo treatments. Using a high-fat diet animal model, we confirmed that LV extracts (LVE) decreased lipogenic metabolism and restored liver function to control levels. To identify active components, we conducted activity-guided fractionation and then isolated compounds. Two compounds, loliolide and pinoresinol, were identified in the dichloromethane fraction, and they significantly attenuated the expression levels of lipogenic factors including sterol regulatory element-binding protein (SREBP)-1, stearoyl-CoA desaturase 1 (SCD1), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC). Importantly, loliolide and pinoresinol significantly accelerated the protein degradation of LXRs by enhanced ubiquitination, which inhibited lipogenesis. These results suggest that loliolide and pinoresinol might be potential candidate supplementary treatments for nonalcoholic fatty liver disease (NAFLD) by reducing lipogenesis through increased ubiquitination of LXRs.


Assuntos
Benzofuranos/administração & dosagem , Furanos/administração & dosagem , Lignanas/administração & dosagem , Lipogênese/efeitos dos fármacos , Receptores X do Fígado/metabolismo , Fígado/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Extratos Vegetais/administração & dosagem , Primulaceae/química , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Humanos , Fígado/metabolismo , Receptores X do Fígado/genética , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos/metabolismo
13.
Microb Cell Fact ; 18(1): 163, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31581944

RESUMO

BACKGROUND: Sustainable production of microbial fatty acids derivatives has the potential to replace petroleum based equivalents in the chemical, cosmetic and pharmaceutical industry. Most fatty acid sources for production oleochemicals are currently plant derived. However, utilization of these crops are associated with land use change and food competition. Microbial oils could be an alternative source of fatty acids, which circumvents the issue with agricultural competition. RESULTS: In this study, we generated a chimeric microbial production system that features aspects of both prokaryotic and eukaryotic fatty acid biosynthetic pathways targeted towards the generation of long chain fatty acids. We redirected the type-II fatty acid biosynthetic pathway of Escherichia coli BL21 (DE3) strain by incorporating two homologues of the beta-ketoacyl-[acyl carrier protein] synthase I and II from the chloroplastic fatty acid biosynthetic pathway of Arabidopsis thaliana. The microbial clones harboring the heterologous pathway yielded 292 mg/g and 220 mg/g DCW for KAS I and KAS II harboring plasmids respectively. Surprisingly, beta-ketoacyl synthases KASI/II isolated from A. thaliana showed compatibility with the FAB pathway in E. coli. CONCLUSION: The efficiency of the heterologous plant enzymes supersedes the overexpression of the native enzyme in the E. coli production system, which leads to cell death in fabF overexpression and fabB deletion mutants. The utilization of our plasmid based system would allow generation of plant like fatty acids in E. coli and their subsequent chemical or enzymatic conversion to high end oleochemical products.


Assuntos
Arabidopsis/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/biossíntese , Engenharia Metabólica , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/síntese química , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/síntese química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Vias Biossintéticas , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Ácido Graxo Sintases/genética , Ácidos Graxos/química , Isoenzimas/síntese química , Isoenzimas/genética , Isoenzimas/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo
14.
Nat Commun ; 10(1): 4055, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492836

RESUMO

Long-chain polyunsaturated fatty acids (LC-PUFAs), particularly the omega-3 LC-PUFAs eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA), have been associated with beneficial health effects. Consequently, sustainable sources have to be developed to meet the increasing demand for these PUFAs. Here, we demonstrate the design and construction of artificial PUFA biosynthetic gene clusters (BGCs) encoding polyketide synthase-like PUFA synthases from myxobacteria adapted for the oleaginous yeast Yarrowia lipolytica. Genomic integration and heterologous expression of unmodified or hybrid PUFA BGCs yielded different yeast strains with specific LC-PUFA production profiles at promising yield and thus valuable for the biotechnological production of distinct PUFAs. Nutrient screening revealed a strong enhancement of PUFA production, when cells were phosphate limited. This represents, to the best of our knowledge, highest concentration of DHA (16.8 %) in total fatty acids among all published PUFA-producing Y. lipolytica strains.


Assuntos
Proteínas de Bactérias/metabolismo , Ácido Graxo Sintases/metabolismo , Ácidos Graxos Insaturados/biossíntese , Myxococcales/enzimologia , Yarrowia/metabolismo , Proteínas de Bactérias/genética , Biotecnologia/métodos , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Graxo Sintases/genética , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Insaturados/metabolismo , Engenharia Metabólica/métodos , Myxococcales/genética , Reprodutibilidade dos Testes
15.
BMC Complement Altern Med ; 19(1): 255, 2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519174

RESUMO

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and is characterized by excessive hepatic lipid accumulation. Many studies have suggested that lipid overload is the key initial factor that contributes to hepatic steatosis. Our previous study indicated that diosgenin (DSG) has a beneficial effect on energy metabolism, but the underlying mechanism remains unclear. METHODS: Human normal hepatocytes (LO2 cells) were incubated with palmitic acid to establish the cell model of nonalcoholic fatty liver. The effects of DSG on lipid metabolism, glucose uptake and mitochondrial function were evaluated. Furthermore, the mechanism of DSG on oxidative stress, lipid consumption and lipid synthesis in LO2 cells was investigated. RESULTS: The results indicated that palmitic acid induced obvious lipid accumulation in LO2 cells and that DSG treatment significantly reduced the intracellular lipid content. DSG treatment upregulated expression of lipolysis proteins, including phospho-AMP activated protein kinase (p-AMPK), phospho-acetyl-coA carboxylase (p-ACC) and carnitine acyl transferase 1A (CPT-1A), and inhibited expression of lipid synthesis-related proteins, including sterol regulatory element-binding protein 1c (SREBP-1c) and fatty acid synthase (FAS). Additionally, DSG-treated cells displayed a marked improvement in mitochondrial function, with less production of reactive oxygen species and a higher mitochondrial membrane potential compared with the model group. CONCLUSION: This study suggests that DSG can reduce intracellular lipid accumulation in LO2 cells and that the underlying mechanism may be related to the improving oxidative stress, increasing fatty acid ß-oxidation and decreasing lipid synthesis. The above changes might be mediated by the activation of the AMPK/ACC/CPT-1A pathway and inhibition of the SREBP-1c/FAS pathway.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/metabolismo , Diosgenina/farmacologia , Ácido Graxo Sintases/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Ácido Palmítico/efeitos adversos , Proteínas Quinases Ativadas por AMP/genética , Acetil-CoA Carboxilase/genética , Carnitina O-Acetiltransferase/genética , Carnitina O-Acetiltransferase/metabolismo , Linhagem Celular , Ácido Graxo Sintases/genética , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/genética , Transdução de Sinais/efeitos dos fármacos , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
16.
Food Funct ; 10(9): 5804-5815, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31461095

RESUMO

Diabetes, an endocrine and metabolic disorder, has become the third most non-infectious chronic disease that threatens human health. Type 2 diabetes (T2D) accounts for more than 90% of diabetic patients, mainly caused by environmental factors. Lactic acid bacteria (LAB) exhibit several health benefits to the host including regulating glucose and lipid metabolism and improving oxidative stress and inflammatory response. However, the anti-diabetic mechanism of probiotics has not been elucidated clearly. In this study, the anti-diabetic effects of Lactobacillus acidophilus KLDS1.1003 and KLDS1.0901 on T2D mice were assessed. Oral administration of L. acidophilus KLDS1.1003 and KLDS1.0901 for 6 weeks significantly improved the epithelial barrier function, which in turn lowered inflammation cytokines, including IL-8, TNF-α and IL-1ß in liver and colon tissue, and prevented liver and colon tissue injuries to some extent. Additionally, L. acidophilus treatment regulated the expression genes that are related to glucose and lipid metabolism. The two tested strains down-regulated the expression of glycogen synthase kinase 3ß (GSK-3ß), fatty acid synthase (FAS) and sterol regulatory element-binding transcription factor 1c (SREBP-1c), and up-regulated the expression of protein kinase B (Akt). However, L. acidophilus KLDS1.0901 is better for improving T2D than L. acidophilus KLDS1.1003. Further research showed that L. acidophilus KLDS1.0901 supplementation could reshape gut microbiota, increasing short chain fatty acid-producing bacteria (Blautia, Roseburia and Anaerotruncus) and the level of SCFAs and decreasing the relative abundance of Gram-negative bacteria such as Desulfovibrio, Alistipes and Bacteroides. Notably, L. acidophilus KLDS1.0901 treatment restored the structure of gut microbiota similar to the control group. These findings suggested that L. acidophilus KLDS1.0901 might be used as a new type of antidiabetic drug candidate.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Microbioma Gastrointestinal/efeitos dos fármacos , Glucose/metabolismo , Hipoglicemiantes/administração & dosagem , Lactobacillus acidophilus/fisiologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Probióticos/administração & dosagem , Animais , Bactérias/classificação , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/microbiologia , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Ácidos Graxos Voláteis/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
17.
Plant J ; 100(6): 1289-1305, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31437318

RESUMO

Chlorella vulgaris is a fast-growing fresh-water microalga cultivated on the industrial scale for applications ranging from food to biofuel production. To advance our understanding of its biology and to establish genetics tools for biotechnological manipulation, we sequenced the nuclear and organelle genomes of Chlorella vulgaris 211/11P by combining next generation sequencing and optical mapping of isolated DNA molecules. This hybrid approach allowed us to assemble the nuclear genome in 14 pseudo-molecules with an N50 of 2.8 Mb and 98.9% of scaffolded genome. The integration of RNA-seq data obtained at two different irradiances of growth (high light, HL versus low light, LL) enabled us to identify 10 724 nuclear genes, coding for 11 082 transcripts. Moreover, 121 and 48 genes, respectively, were found in the chloroplast and mitochondrial genome. Functional annotation and expression analysis of nuclear, chloroplast and mitochondrial genome sequences revealed particular features of Chlorella vulgaris. Evidence of horizontal gene transfers from chloroplast to mitochondrial genome was observed. Furthermore, comparative transcriptomic analyses of LL versus HL provided insights into the molecular basis for metabolic rearrangement under HL versus LL conditions leading to enhanced de novo fatty acid biosynthesis and triacylglycerol accumulation. The occurrence of a cytosolic fatty acid biosynthetic pathway could be predicted and its upregulation upon HL exposure was observed, consistent with the increased lipid amount under HL conditions. These data provide a rich genetic resource for future genome editing studies, and potential targets for biotechnological manipulation of Chlorella vulgaris or other microalgae species to improve biomass and lipid productivity.


Assuntos
Aclimatação/genética , Aclimatação/efeitos da radiação , Chlorella vulgaris/genética , Chlorella vulgaris/metabolismo , Chlorella vulgaris/efeitos da radiação , Luz , Anotação de Sequência Molecular , Sequência de Bases , Biocombustíveis , Biomassa , Vias Biossintéticas/genética , Vias Biossintéticas/fisiologia , Vias Biossintéticas/efeitos da radiação , Biotecnologia , Chlorella vulgaris/crescimento & desenvolvimento , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/biossíntese , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Ontologia Genética , Transferência Genética Horizontal , Genoma Mitocondrial , Genoma de Planta , Lipídeos/biossíntese , Meiose , Filogenia , Transcriptoma , Triglicerídeos/biossíntese
18.
Insect Biochem Mol Biol ; 112: 103203, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31425851

RESUMO

Insect cuticular hydrocarbons (CHCs), the evolutionary products of aquatic hexapod ancestors expanding to terrestrial environment, are deposited on the surface of insect integument and originally functioned primarily as waterproofing agents. CHCs are derived from the conserved fatty acid synthesis pathway in insects. However, the pivotal fatty acid synthase (FAS) involved in hydrocarbon (HC) biosynthesis remains unknown in many insect orders including the primitive Blattodea. Here, we investigated functional FAS genes that modulate cuticular lipid biogenesis in the German cockroach, Blattella germanica (L.). Based on our full-length transcriptomic data and the available genomic data, seven FAS genes (BgFas1-7) were identified from B. germanica. Tissue-specific expression analysis revealed that BgFas1, BgFas3, BgFas4 and BgFas7 were highly expressed in the integument, whereas BgFas2 was dominantly expressed in the fat body. BgFas5/6 mRNA was almost negligible in the tested tissues. Systemic RNAi screen was performed against BgFas1-7, we found that only RNAi knockdown of BgFas1 caused a dramatic reduction of methyl-branched HCs (mbHCs) and a slight decrease of straight-chain HCs (scHCs) for both internal and external HCs. Significant reduction of cuticular free fatty acids (cFFAs) was also detected within BgFas1-repressed cockroaches, while repression of CYP4G19 resulted in dramatic increase of cFFAs. Moreover, we found that BgFas1 mRNA levels were correlated with insect molting cycles, and could be induced by long-term mild dryness treatment. Furthermore, desiccation assay revealed that BgFas1 suppression accelerated water loss and led to early death of cockroaches under desiccation. Our results indicate that BgFas1 is necessary for both HC and cFFA biosynthesis in B. germanica. In addition, our study also confirms that cuticular lipids, particularly mbCHCs, are critical for desiccation resistance in B. germanica.


Assuntos
Blattellidae/enzimologia , Ácido Graxo Sintases/genética , Hidrocarbonetos/metabolismo , Animais , Blattellidae/genética , Ácidos Graxos/biossíntese , Genes de Insetos , Fenômenos Fisiológicos do Tegumento Comum , Interferência de RNA
19.
Sci Rep ; 9(1): 10502, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324846

RESUMO

The peroxisomal ABC transporter, Comatose (CTS), a full length transporter from Arabidopsis has intrinsic acyl-CoA thioesterase (ACOT) activity, important for physiological function. We used molecular modelling, mutagenesis and biochemical analysis to identify amino acid residues important for ACOT activity. D863, Q864 and T867 lie within transmembrane helix 9. These residues are orientated such that they might plausibly contribute to a catalytic triad similar to type II Hotdog fold thioesterases. When expressed in Saccharomyces cerevisiae, mutation of these residues to alanine resulted in defective of ß-oxidation. All CTS mutants were expressed and targeted to peroxisomes and retained substrate-stimulated ATPase activity. When expressed in insect cell membranes, Q864A and S810N had similar ATPase activity to wild type but greatly reduced ACOT activity, whereas the Walker A mutant K487A had greatly reduced ATPase and no ATP-dependent ACOT activity. In wild type CTS, ATPase but not ACOT was stimulated by non-cleavable C14 ether-CoA. ACOT activity was stimulated by ATP but not by non-hydrolysable AMPPNP. Thus, ACOT activity depends on functional ATPase activity but not vice versa, and these two activities can be separated by mutagenesis. Whether D863, Q864 and T867 have a catalytic role or play a more indirect role in NBD-TMD communication is discussed.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Ácido Graxo Sintases/metabolismo , Tioléster Hidrolases/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Domínio Catalítico , Linhagem Celular , Ácido Graxo Sintases/genética , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Ácido Oleico/metabolismo , Oxirredução , Peroxissomos/enzimologia , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Spodoptera , Relação Estrutura-Atividade , Tioléster Hidrolases/genética
20.
Lipids Health Dis ; 18(1): 135, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31174532

RESUMO

BACKGROUND: Heat induced by infrared (IR) radiation from sun exposure increases skin temperature and can lead to thermal and photo-aging. However, little is known about the relationship between heat induced by IR radiation and lipid biosynthesis in human sebocytes. This study investigated the expression of factors involved in lipid biosynthesis in human sebocytes exposed to heat. The effect of Cassia tora extract and chrysophanol, which is widely used as anti-inflammatory agent, on the heat shock effect in sebocytes was then examined. METHODS: For the treatment, cells were maintained in culture medium without FBS (i.e., serum starved) for 6 h and then moved for 30 min to incubators at 37 °C (control), 41 °C, or 44 °C (heat shock). Culture media were replaced with fresh media without FBS. To investigate expression of gene and signaling pathway, we performed western blotting. Lipid levels were assessed by Nile red staining. The cytokine levels were measured by cytokine array and ELISA kit. RESULTS: We found that peroxisome proliferator-activated receptor (PPAR)γ and fatty acid synthase (FAS) were upregulated and the c-Jun N-terminal kinase (JNK)/p38 signaling pathways were activated in human sebocytes following heat exposure. Treatment with Cassia tora seed extract and chrysophanol suppressed this up-regulation of PPARγ and FAS and also suppressed the increase in IL-1ß levels. CONCLUSION: These findings provide evidence that IR radiation can stimulate sebum production; Cassia tora seed extract and chrysophanol can reverse lipid stimulated inflammatory mediation, and may therefore be useful for treating skin disorders such as acne vulgaris.


Assuntos
Antraquinonas/farmacologia , Cassia/química , Lipogênese/efeitos dos fármacos , Extratos Vegetais/farmacologia , Antraquinonas/química , Células Epiteliais/química , Ácido Graxo Sintases/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Temperatura Alta/efeitos adversos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Lipogênese/genética , Lipogênese/efeitos da radiação , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos da radiação , PPAR gama/genética , Extratos Vegetais/química , Radiação , Transdução de Sinais/efeitos dos fármacos , Temperatura Cutânea/efeitos da radiação , Proteínas Quinases p38 Ativadas por Mitógeno/genética
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