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1.
Nat Commun ; 12(1): 1051, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594068

RESUMO

In metabolic engineering, loss-of-function experiments are used to understand and optimise metabolism. A conditional gene inactivation tool is required when gene deletion is lethal or detrimental to growth. Here, we exploit auxin-inducible protein degradation as a metabolic engineering approach in yeast. We demonstrate its effectiveness using terpenoid production. First, we target an essential prenyl-pyrophosphate metabolism protein, farnesyl pyrophosphate synthase (Erg20p). Degradation successfully redirects metabolic flux toward monoterpene (C10) production. Second, depleting hexokinase-2, a key protein in glucose signalling transduction, lifts glucose repression and boosts production of sesquiterpene (C15) nerolidol to 3.5 g L-1 in flask cultivation. Third, depleting acetyl-CoA carboxylase (Acc1p), another essential protein, delivers growth arrest without diminishing production capacity in nerolidol-producing yeast, providing a strategy to decouple growth and production. These studies demonstrate auxin-mediated protein degradation as an advanced tool for metabolic engineering. It also has potential for broader metabolic perturbation studies to better understand metabolism.


Assuntos
Ácidos Indolacéticos/farmacologia , Engenharia Metabólica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Terpenos/metabolismo , Proteínas de Bactérias/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Coenzima A Ligases/metabolismo , Glucose/metabolismo , Hexoquinase/metabolismo , Limoneno/metabolismo , Análise do Fluxo Metabólico , Fosfatos de Poli-Isoprenil/metabolismo , Proteólise/efeitos dos fármacos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sesquiterpenos/metabolismo
2.
Biochem Biophys Res Commun ; 545: 81-88, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33548628

RESUMO

Cervical cancer remains the leading cause of cancerous death among women worldwide. Oleanolic acid (OA) is a substance that occurs naturally in the leaves, fruits, and rhizomes of plants and has anti-cancer activity. In this study, tumor-bearing mice were used as the animal model and Hela cells were used as cellular model. In vivo experiments have showed that OA significantly reduced the size and mass of cervical cancer tumors in mice. In vitro experiments have showed that OA significantly reduced the viability and proliferative capacity of Hela cells. In both in vivo and in vitro assays, OA increased the oxidative stress levels and Fe2+ content, and increased the expression of ferroptosis-related proteins. We found that ACSL4 was highly expressed in both xenograft models and cervical carcinoma cells with OA treatment. Further use of siRNA to interfere with ACSL4 expression in cervical cancer cells revealed that the inhibitory effect of OA on cell viability and proliferative capacity was counteracted, while a decrease in ROS levels and GPX4 was detected, suggesting that OA activated ferroptosis in Hela cells by promoting ACSL4 expression, thereby reducing the survival rate of Hela cells. Therefore, promotion of ACSL4-dependent ferroptosis by OA may be a potential approach for the treatment of cervical cancer.


Assuntos
Coenzima A Ligases/metabolismo , Ácido Oleanólico/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Coenzima A Ligases/antagonistas & inibidores , Coenzima A Ligases/genética , Feminino , Ferroptose/efeitos dos fármacos , Células HeLa , Humanos , Ferro/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Plant Physiol Biochem ; 161: 1-11, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33556720

RESUMO

In plants, Long-chain acyl-CoA synthetases (LACSs) play key roles in activating fatty acids to fatty acyl-CoA thioesters, which are then further involved in lipid synthesis and fatty acid catabolism. LACSs have been intensively studied in Arabidopsis, but its evolutionary relationship in green plants is unexplored. In this study, we performed a comprehensive genome-wide analysis of the LACS gene family across green plants followed by phylogenetic clustering analysis, gene structure determination, detection of conserved motifs, gene expression in tissues and subcellular localization. Our results identified LACS genes in 122 plant species including algae, low land plants (i.e., mosses and lycophytes), monocots, and eudicots. In total, 697 sequences were identified, and 629 sequences were selected because of alignment and some duplication errors. The retrieved amino acid sequences ranged from 271 to 1056 residues and diversified in intron/exon patterns in different LACSs. Phylogenetic clustering grouped LACS gene family into six major clades with distinct potential functions. This classification is well supported by examining gene structure and conserved motifs. Also, gene expression analysis and subcellular localization substantiate with clade division in the phylogeny, indicating that the evolutionary pattern is visible in their functionality. Additionally, experimental analysis of lacs2 mutant validated that LACS2 plays key roles in suberin synthesis. Thus, our study not only provides an evolutionary mechanism underlying functional diversification but also lays the foundation for further elucidation of the LACS gene family.


Assuntos
Arabidopsis , Regulação da Expressão Gênica de Plantas , Arabidopsis/genética , Arabidopsis/metabolismo , Coenzima A , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Perfilação da Expressão Gênica , Filogenia
4.
Arch Biochem Biophys ; 700: 108773, 2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33485846

RESUMO

Fatty acids are essential cellular building blocks and a major energy source. Regardless of their metabolic fate, fatty acids first need to be activated by forming a thioester with a coenzyme A group. This reaction is carried out by acyl-CoA synthetases (ACSs), of which ACSL1 (long-chain acyl-CoA synthetase 1) is an important member. Two bacterial homologues of ACSL1 crystal structures have been solved previously. One is a soluble dimeric protein, and the other is a monomeric peripheral membrane protein. The mammalian ACSL1 is a membrane protein with an N-terminal transmembrane helix. To characterize the mammalian ACSL1, we purified the full-length mouse ACSL1 and reconstituted it into lipid nanodiscs. Using enzymatic assays, mutational analysis, and cryo-electron microscopy, we show that mouse ACSL1 is active as a monomer.


Assuntos
Coenzima A Ligases/química , Animais , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Cristalografia por Raios X , Camundongos , Multimerização Proteica , Estrutura Secundária de Proteína
5.
Int J Mol Sci ; 22(1)2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33401455

RESUMO

The current genome editing system Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR/Cas9) has already confirmed its proficiency, adaptability, and simplicity in several plant-based applications. Together with the availability of a vast amount of genome data and transcriptome data, CRISPR/Cas9 presents a massive opportunity for plant breeders and researchers. The successful delivery of ribonucleoproteins (RNPs), which are composed of Cas9 enzyme and a synthetically designed single guide RNA (sgRNA) and are used in combination with various transformation methods or lately available novel nanoparticle-based delivery approaches, allows targeted mutagenesis in plants species. Even though this editing technique is limitless, it has still not been employed in many plant species to date. Chickpea is the second most crucial winter grain crop cultivated worldwide; there are currently no reports on CRISPR/Cas9 gene editing in chickpea. Here, we selected the 4-coumarate ligase (4CL) and Reveille 7 (RVE7) genes, both associated with drought tolerance for CRISPR/Cas9 editing in chickpea protoplast. The 4CL represents a key enzyme involved in phenylpropanoid metabolism in the lignin biosynthesis pathway. It regulates the accumulation of lignin under stress conditions in several plants. The RVE7 is a MYB transcription factor which is part of regulating circadian rhythm in plants. The knockout of these selected genes in the chickpea protoplast using DNA-free CRISPR/Cas9 editing represents a novel approach for achieving targeted mutagenesis in chickpea. Results showed high-efficiency editing was achieved for RVE7 gene in vivo compared to the 4CL gene. This study will help unravel the role of these genes under drought stress and understand the complex drought stress mechanism pathways. This is the first study in chickpea protoplast utilizing CRISPR/Cas9 DNA free gene editing of drought tolerance associated genes.


Assuntos
Proteína 9 Associada à CRISPR , Cicer/genética , Coenzima A Ligases/genética , Edição de Genes/métodos , Estresse Fisiológico , Fatores de Transcrição/genética , Cicer/enzimologia , Cicer/metabolismo , Cicer/fisiologia , Coenzima A Ligases/metabolismo , Coenzima A Ligases/fisiologia , Secas , Técnicas de Inativação de Genes , Lignina/biossíntese , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia
6.
Gene ; 764: 145094, 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-32860898

RESUMO

Long chain acyl-CoA synthetases (ACSLs), which drive the conversion of long chain fatty acid into acyl-CoA, an ingredient of lipid synthesis, have been well-acknowledged to exert an indispensable role in many metabolic processes in mammals, especially lipid metabolism. However, in chicken, the evolutionary characteristics, expression profiles and regulatory mechanisms of ACSL gene family are rarely understood. Here, we analyzed the genomic synteny, gene structure, evolutionary event and functional domains of the ACSL gene family members using bioinformatics methods. The spatiotemporal expression profiles of ACSL gene family, and their regulatory mechanism were investigated via bioinformatics analysis incorporated with in vivo and in vitro estrogen-treated experiments. Our results indicated that ACSL2 gene was indeed evolutionarily lost in the genome of chicken. Chicken ACSLs shared an AMP-binding functional domain, as well as highly conversed ATP/AMP and FACS signature motifs, and were clustered into two clades, ACSL1/5/6 and ACSL3/4, based on high sequence similarity, similar gene features and conversed motifs. Chicken ACSLs showed differential tissue expression distributions, wherein the significantly decreased expression level of ACSL1 and the significantly increased expression level of ACSL5 were found, respectively, the expression levels of the other ACSL members remained unchanged in the liver of peak-laying hens versus pre-laying hens. Moreover, the transcription activity of ACSL1, ACSL3 and ACSL4 was silenced and ACSL6 was activated by estrogen, but no response to ACSL5. In conclusion, though having highly conversed functional domains, chicken ACSL gene family is organized into two separate groups, ACSL1/5/6 and ACSL3/4, and exhibits varying expression profiles and estrogen effects. These results not only pave the way for better understanding the specific functions of ACSL genes in avian lipid metabolism, but also provide a valuable evidence for gene family characteristics.


Assuntos
Galinhas/genética , Coenzima A Ligases/genética , Evolução Molecular , Metabolismo dos Lipídeos/genética , Família Multigênica/genética , Acil Coenzima A/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Galinhas/crescimento & desenvolvimento , Galinhas/metabolismo , Coenzima A Ligases/metabolismo , Biologia Computacional , Estrogênios/metabolismo , Ácidos Graxos/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hepatócitos , Cultura Primária de Células , Domínios Proteicos/genética , Análise Espaço-Temporal , Sintenia
7.
Biochem Biophys Res Commun ; 536: 100-106, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33373853

RESUMO

In regulated cell death, genetically encoded molecular machinery destroys cells. This process is not only essential for organ development and homeostasis, but also leads to pathological diseases. One form of regulated cell death is ferroptosis, which is an iron-dependent oxidative cell death caused by lipid peroxidation. Although inducing ferroptosis is an emerging anticancer strategy, the molecular mechanism underlying tumor resistance to ferroptotic cell death is still unclear. Here, we show that pirin (PIR), an iron-binding nuclear protein, plays a previously unrecognized role in mediating ferroptosis resistance in human pancreatic cancer cells. The transcription factor NFE2L2 mediates the upregulation of PIR during ferroptosis caused by small-molecule compounds (e.g., erastin or RSL3). PIR is a nuclear redox sensor and regulator, and increasing it limits the oxidative damage of DNA and the subsequent cytoplasmic transport and extracellular release of HMGB1. In contrast, the depletion of PIR initiates HMGB1-dependent autophagy by binding to BECN1, and subsequently promotes ferroptosis by activating ACSL4. Consequently, in cell cultures and xenograft mouse models, blocking PIR signaling enhances ferroptosis-mediated tumor growth suppression. Together, these findings provide new insights into the molecular mechanisms of autophagy-dependent ferroptosis.


Assuntos
Autofagia , Núcleo Celular/metabolismo , Dioxigenases/metabolismo , Ferroptose , Animais , Linhagem Celular Tumoral , Coenzima A Ligases/metabolismo , Dioxigenases/genética , Proteína HMGB1/metabolismo , Humanos , Camundongos Nus , Fator 2 Relacionado a NF-E2/metabolismo , Oxirredução , Regulação para Cima/genética
8.
Nature ; 585(7823): 113-118, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32814895

RESUMO

Cancer cells, including melanoma cells, often metastasize regionally through the lymphatic system before metastasizing systemically through the blood1-4; however, the reason for this is unclear. Here we show that melanoma cells in lymph experience less oxidative stress and form more metastases than melanoma cells in blood. Immunocompromised mice with melanomas derived from patients, and immunocompetent mice with mouse melanomas, had more melanoma cells per microlitre in tumour-draining lymph than in tumour-draining blood. Cells that metastasized through blood, but not those that metastasized through lymph, became dependent on the ferroptosis inhibitor GPX4. Cells that were pretreated with chemical ferroptosis inhibitors formed more metastases than untreated cells after intravenous, but not intralymphatic, injection. We observed multiple differences between lymph fluid and blood plasma that may contribute to decreased oxidative stress and ferroptosis in lymph, including higher levels of glutathione and oleic acid and less free iron in lymph. Oleic acid protected melanoma cells from ferroptosis in an Acsl3-dependent manner and increased their capacity to form metastatic tumours. Melanoma cells from lymph nodes were more resistant to ferroptosis and formed more metastases after intravenous injection than did melanoma cells from subcutaneous tumours. Exposure to the lymphatic environment thus protects melanoma cells from ferroptosis and increases their ability to survive during subsequent metastasis through the blood.


Assuntos
Ferroptose , Linfa/metabolismo , Melanoma/patologia , Metástase Neoplásica/patologia , Animais , Sobrevivência Celular , Coenzima A Ligases/metabolismo , Feminino , Ferroptose/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Ferro/metabolismo , Masculino , Melanoma/sangue , Melanoma/metabolismo , Camundongos , Metástase Neoplásica/tratamento farmacológico , Ácido Oleico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Análise de Componente Principal
9.
Nat Commun ; 11(1): 3941, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770005

RESUMO

Anaerobic oxidation of methane (AOM) mediated by anaerobic methanotrophic archaea (ANME) is the primary process that provides energy to cold seep ecosystems by converting methane into inorganic carbon. Notably, cold seep ecosystems are dominated by highly divergent heterotrophic microorganisms. The role of the AOM process in supporting heterotrophic population remains unknown. We investigate the acetogenic capacity of ANME-2a in a simulated cold seep ecosystem using high-pressure biotechnology, where both AOM activity and acetate production are detected. The production of acetate from methane is confirmed by isotope-labeling experiments. A complete archaeal acetogenesis pathway is identified in the ANME-2a genome, and apparent acetogenic activity of the key enzymes ADP-forming acetate-CoA ligase and acetyl-CoA synthetase is demonstrated. Here, we propose a modified model of carbon cycling in cold seeps: during AOM process, methane can be converted into organic carbon, such as acetate, which further fuels the heterotrophic community in the ecosystem.


Assuntos
Acetatos/metabolismo , Archaea/enzimologia , Proteínas de Bactérias/metabolismo , Coenzima A Ligases/metabolismo , Metano/metabolismo , Anaerobiose , Archaea/genética , Proteínas de Bactérias/genética , Ciclo do Carbono/fisiologia , Coenzima A Ligases/genética , Genoma Arqueal , Sedimentos Geológicos/microbiologia , Redes e Vias Metabólicas/genética , Oxirredução , Água do Mar/microbiologia
10.
Am J Physiol Renal Physiol ; 319(4): F603-F611, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32830538

RESUMO

The acyl-CoA synthetase medium-chain family member 2 (Acsm2) gene was first identified and cloned by our group as a kidney-specific "KS" gene. However, its expression pattern and function remain to be clarified. In the present study, we found that the Acsm2 gene was expressed specifically and at a high level in normal adult kidneys. Expression of Acsm2 in kidneys followed a maturational pattern: it was low in newborn mice and increased with kidney development and maturation. In situ hybridization and immunohistochemistry revealed that Acsm2 was expressed specifically in proximal tubular cells of adult kidneys. Data from the Encyclopedia of DNA Elements database revealed that the Acsm2 gene locus in the mouse has specific histone modifications related to the active transcription of the gene exclusively in kidney cells. Following acute kidney injury, partial unilateral ureteral obstruction, and chronic kidney diseases, expression of Acsm2 in the proximal tubules was significantly decreased. In human samples, the expression pattern of ACSM2A, a homolog of mouse Acsm2, was similar to that in mice, and its expression decreased with several types of renal injuries. These results indicate that the expression of Acsm2 parallels the structural and functional maturation of proximal tubular cells. Downregulation of its expression in several models of kidney disease suggests that Acms2 may serve as a novel marker of proximal tubular injury and/or dysfunction.


Assuntos
Coenzima A Ligases/metabolismo , Células Epiteliais/metabolismo , Túbulos Renais Proximais/metabolismo , Lesão Renal Aguda/enzimologia , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Animais , Coenzima A Ligases/genética , Modelos Animais de Doenças , Células Epiteliais/patologia , Fibrose , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Insuficiência Renal Crônica/enzimologia , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Renina/genética , Renina/metabolismo , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia
11.
DNA Cell Biol ; 39(9): 1573-1582, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32678986

RESUMO

Many immune cells participate in the pathogenesis of ulcerative colitis (UC), and fatty acid metabolism (FAM) is reported to supporting their cell-specific functions and proliferation, but the underlying mechanism is unclear. This study aimed to investigate the relationship between FAM and inflammation in colon tissues and identify potential therapeutic targets for regulating immune response. A total of 870 different expression genes (DEGs), 304 immunity-related DEGs, and 11 FAM-related DEGs were obtained, gene ontology analysis results showed that immune DEGs were significantly enriched in neutrophil migration, positive regulation of T cell activation. Fifteen types of immune cells were identified in inflamed colon tissues. Five FAM-related DEGs (ACOX1, ACSL4, ELOVL5, FADS2, and SCD) were highly correlated with immunity-related DEGs, and ACSL4, ELOVL5, and FADS2 were significantly upregulated in immune cells, while SCD is downregulated. Five FAM-related DEGs were highly correlated with immune cells. The study promotes the understanding of the pathogenesis of FAM in UC immune cells.


Assuntos
Colite Ulcerativa/genética , Ácidos Graxos/metabolismo , Redes Reguladoras de Genes , Transcriptoma , Acil-CoA Oxidase/genética , Acil-CoA Oxidase/metabolismo , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Colite Ulcerativa/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Elongases de Ácidos Graxos/genética , Elongases de Ácidos Graxos/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Linfócitos/metabolismo , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo
12.
Artigo em Inglês | MEDLINE | ID: mdl-32126286

RESUMO

Alterations in fatty acid metabolism are associated with impaired glucose uptake in skeletal muscle. Long-chain acyl-CoA synthetase (Acsl) 6 is the one of the Acsl isoforms expressed in skeletal muscle although its role in muscle energy metabolism has not been studied. Thus, the aims of this study were to investigate the role of Acsl6 in fatty acid partitioning and glucose uptake in differentiated skeletal myotubes using a siRNA-mediated knockdown approach. Compared with cells transfected with control siRNA, cells transfected with Acsl6 siRNA exhibited reduced intracellular triacylglycerol (TAG) accumulation. The initial rate of [1­14C]­oleic acid uptake was not altered while the incorporation of [1­14C]­acetic acids into total cellular lipids decreased under Acsl6 knockdown (p < 0.05). In a metabolic labeling study, Acsl6 suppression decreased the incorporation of [1­14C]­oleic acids and [1­14C]­acetic acids into TAG and diacylglycerol (DAG) (p < 0.05). During the chase period of a pulse-chase experiment, Acsl6 suppression increased the intracellular free fatty acids and decreased the fatty acid channeling toward the reacylation of TAG (p < 0.05). The incorporation of the labeled fatty acids into acid-soluble metabolites, ß-oxidation product, was not changed under Acsl6 knockdown. Acsl6 siRNA decreased the insulin-induced uptake of [1­14C]­2­deoxyglucose (p < 0.05) but did not change the glucose uptake in the presence of acipimox, inhibitor of lipolysis. Suppression of Acsl6 deteriorated Akt phosphorylation and Glut4 mRNA expression in response to insulin. These results suggest that Acsl6 activates and channels fatty acids toward anabolic pathways and has a role in glucose and fatty acid cycling through the re-esterification of fatty acids in skeletal muscle.


Assuntos
Coenzima A Ligases/metabolismo , Ácidos Graxos/metabolismo , Glucose/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Animais , Linhagem Celular , Coenzima A Ligases/genética , Diglicerídeos/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Insulina/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Triglicerídeos/metabolismo
13.
Appl Environ Microbiol ; 86(10)2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32144106

RESUMO

In Lysobacter enzymogenes OH11, RpfB1 and RpfB2 were predicted to encode acyl coenzyme A (CoA) ligases. RpfB1 is located in the Rpf gene cluster. Interestingly, we found an RpfB1 homolog (RpfB2) outside this canonical gene cluster, and nothing is known about its functionality or mechanism. Here, we report that rpfB1 and rpfB2 can functionally replace EcFadD in the Escherichia coli fadD mutant JW1794. RpfB activates long-chain fatty acids (n-C16:0 and n-C18:0) for the corresponding fatty acyl-CoA ligase (FCL) activity in vitro, and Glu-361 plays critical roles in the catalytic mechanism of RpfB1 and RpfB2. Deletion of rpfB1 and rpfB2 resulted in significantly increased heat-stable antifungal factor (HSAF) production, and overexpression of rpfB1 or rpfB2 completely suppressed HSAF production. Deletion of rpfB1 and rpfB2 resulted in increased L. enzymogenes diffusible signaling factor 3 (LeDSF3) synthesis in L. enzymogenes Overall, our results showed that changes in intracellular free fatty acid levels significantly altered HSAF production. Our report shows that intracellular free fatty acids are required for HSAF production and that RpfB affects HSAF production via FCL activity. The global transcriptional regulator Clp directly regulated the expression of rpfB1 and rpfB2 In conclusion, these findings reveal new roles of RpfB in antibiotic biosynthesis in L. enzymogenes IMPORTANCE Understanding the biosynthetic and regulatory mechanisms of heat-stable antifungal factor (HSAF) could improve the yield in Lysobacter enzymogenes Here, we report that RpfB1 and RpfB2 encode acyl coenzyme A (CoA) ligases. Our research shows that RpfB1 and RpfB2 affect free fatty acid metabolism via fatty acyl-CoA ligase (FCL) activity to reduce the substrate for HSAF synthesis and, thereby, block HSAF production in L. enzymogenes Furthermore, these findings reveal new roles for the fatty acyl-CoA ligases RpfB1 and RpfB2 in antibiotic biosynthesis in L. enzymogenes Importantly, the novelty of this work is the finding that RpfB2 lies outside the Rpf gene cluster and plays a key role in HSAF production, which has not been reported in other diffusible signaling factor (DSF)/Rpf-producing bacteria.


Assuntos
Antifúngicos/metabolismo , Proteínas de Bactérias/genética , Coenzima A Ligases/genética , Lysobacter/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Coenzima A Ligases/química , Coenzima A Ligases/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Lysobacter/metabolismo , Oxirredução , Alinhamento de Sequência
14.
Mol Cell Biochem ; 468(1-2): 129-142, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32185674

RESUMO

Fibrosis process in the liver is a clinical condition established in response to chronic lesions and may be reversible in many situations. In this process, hepatic stellate cells (HSCs) activate and produce extracellular matrix compounds. During fibrosis, the lipid metabolism is also altered and contributes to the transdifferentiation of the HSCs. Thus, controlling lipid metabolism in HSCs is suggested as a method to control or reverse the fibrotic condition. In the search for therapies that modulate lipid metabolism and treat liver diseases, silymarin has been identified as a relevant natural compound to treat liver pathologies. The present study aimed to evaluate the cellular and molecular effects of silymarin in the transdifferentiation process of HSCs (LX-2) from activated phenotype to a more quiesced-like cells , also focusing on understanding the modulatory effects of silymarin on lipid metabolism of HSCs. In our analyses, 100 µM of silymarin reduced the synthesis of actin filaments in activated cells, the synthesis of the protein level of α-SMA, and other pro-fibrotic factors such as CTGF and PFGF. The concentration of 150 µM silymarin did not reverse the activation aspects of LX-2 cells. However, both evaluated concentrations of the natural compound protected the cells from the negative effects of dimethyl sulfoxide (DMSO). Furthermore, we evaluated lipid-related molecules correlated to the transdifferentiation process of LX-2, and 100 µM of silymarin demonstrated to control molecules associated with lipid metabolism such as FASN, MLYCD, ACSL4, CPTs, among others. In contrast, cellular incubation with 150 µM of silymarin increased the synthesis of long-chain fatty acids and triglycerides, regarding the higher presence of DMSO (v/v) in the solvent. In conclusion, silymarin acts as a hepatoprotective agent and modulates the pro-fibrogenic stimuli of LX-2 cells, whose effects depend on stress levels in the cellular environment.


Assuntos
Transdiferenciação Celular/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Cirrose Hepática/metabolismo , Substâncias Protetoras/farmacologia , Silimarina/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Linhagem Celular , Cromatografia Gasosa , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Dimetil Sulfóxido/toxicidade , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Células Estreladas do Fígado/enzimologia , Células Estreladas do Fígado/metabolismo , Humanos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/genética , Espectrometria de Massas , Triglicerídeos/metabolismo
15.
Oncogene ; 39(14): 2948-2960, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32034305

RESUMO

Enhanced prostaglandin production promotes the development and progression of cancer. Prostaglandins are generated from arachidonic acid (AA) by the action of cyclooxygenase (COX) isoenzymes. However, how cancer cells are able to maintain an elevated supply of AA for prostaglandin production remains unclear. Here, by using lung cancer cell lines and clinically relevant KrasG12D-driven mouse models, we show that the long-chain acyl-CoA synthetase (ACSL3) channels AA into phosphatidylinositols to provide the lysophosphatidylinositol-acyltransferase 1 (LPIAT1) with a pool of AA to sustain high prostaglandin synthesis. LPIAT1 knockdown suppresses proliferation and anchorage-independent growth of lung cancer cell lines, and hinders in vivo tumorigenesis. In primary human lung tumors, the expression of LPIAT1 is elevated compared with healthy tissue, and predicts poor patient survival. This study uncovers the ACSL3-LPIAT1 axis as a requirement for the sustained prostaglandin synthesis in lung cancer with potential therapeutic value.


Assuntos
Aciltransferases/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Coenzima A Ligases/metabolismo , Prostaglandinas/metabolismo , Transdução de Sinais/fisiologia , Células A549 , Animais , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares , Masculino , Camundongos , Camundongos Endogâmicos NOD
16.
Am J Pathol ; 190(4): 830-843, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32035059

RESUMO

The molecular mechanisms of prostate inflammation are unclear. We hypothesized that heme oxygenase 1 (HMOX1; HO-1), an enzyme responsible for degradation of heme to carbon monoxide, bilirubin, and iron, is an important regulator of inflammation and epithelial responses in the prostate. Injection of non-uropathogenic Escherichia coli (MG1655 strain) or phosphate-buffered saline into the urethra of mice led to increased numbers of CD45+ leukocytes and mitotic markers (phosphorylated histone H3 and phosphorylated ERK1/2) in the prostate glands. Leukocyte infiltration was elevated in the prostates harvested from mice lacking HO-1 in myeloid compartment. Conversely, exogenous carbon monoxide (250 ppm) increased IL-1ß levels and suppressed cell proliferation in the prostates. Carbon monoxide did not affect the number of infiltrating CD45+ cells in the prostates of E. coli- or phosphate-buffered saline-treated mice. Interestingly, immunomodulatory effects of HO-1 and/or carbon monoxide correlated with early induction of the long-chain acyl-CoA synthetase 1 (ACSL1). ACSL1 levels were elevated in response to E. coli treatment, and macrophage-expressed ACSL1 was in part required for controlling of IL-1ß expression and prostate cancer cell colony growth in soft agar. These results suggest that HO-1 and/or carbon monoxide might play a distinctive role in modulating prostate inflammation, cell proliferation, and IL-1ß levels in part via an ACSL1-mediated pathway.


Assuntos
Infecções por Escherichia coli/complicações , Heme Oxigenase-1/metabolismo , Heme/metabolismo , Inflamação/imunologia , Metabolismo dos Lipídeos/imunologia , Proteínas de Membrana/metabolismo , Próstata/imunologia , Animais , Bilirrubina/metabolismo , Monóxido de Carbono/metabolismo , Proliferação de Células , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Heme Oxigenase-1/genética , Inflamação/metabolismo , Inflamação/microbiologia , Inflamação/patologia , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Próstata/metabolismo , Próstata/microbiologia , Próstata/patologia , Transdução de Sinais
17.
Int J Mol Sci ; 21(5)2020 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-32106618

RESUMO

Echinochloa crus-galli var. mitis has rarely been reported for herbicide resistance, and no case of quinclorac resistance has been reported so far. Synthetic auxin-type herbicide quinclorac is used extensively to control rice weeds worldwide. A long history of using quinclorac in Chinese rice fields escalated the resistance in E. crus-galli var. mitis against this herbicide. Bioassays in Petri plates and pots exhibited four biotypes that evolved into resistance to quinclorac ranking as JS01-R > AH01-R > JS02-R > JX01-R from three provinces of China. Ethylene production in these biotypes was negatively correlated with resistance level and positively correlated with growth inhibition. Determination of the related ethylene response pathway exhibited resistance in biotypes that recorded a decline in 1-aminocyclopropane-1-carboxylic acid (ACC) content, ACC synthase oxidase activities, and less inducible ACS and ACO genes expressions than the susceptible biotype, suggesting that there was a positive correlation between quinclorac resistance and ethylene biosynthesis inhibition. Cyanides produced during the ethylene biosynthesis pathway mainly degraded by the activity of ß-cyanoalanine synthase (ß-CAS). Resistant biotypes exhibited higher ß-CAS activity than the susceptible ones. Nucleotide changes were found in the EcCAS gene of resistant biotypes as compared to sensitive ones that caused three amino acid substitutions (Asn-105-Lys, Gln-195-Glu, and Gly-298-Val), resulting in alteration of enzyme structure, increased binding residues in the active site with its cofactor, and decreased binding free energy; hence, its activity was higher in resistant biotypes. Moreover, these mutations increased the structural stability of the enzyme. In view of the positive correlation between ethylene biosynthesis inhibition and cyanide degradation with resistance level, it is concluded that the alteration in ethylene response pathway or at least variation in ACC synthase and ACC oxidase enzyme activities-due to less relative expression of ACS and ACO genes and enhanced ß-CAS activity, as well as mutation and increased relative expression of EcCAS gene-can be considered as a probable mechanism of quinclorac resistance in E. crus-galli var. mitis.


Assuntos
Cianetos/metabolismo , Echinochloa/genética , Etilenos/biossíntese , Resistência a Herbicidas , Herbicidas/toxicidade , Quinolinas/toxicidade , Substituição de Aminoácidos , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Echinochloa/efeitos dos fármacos , Echinochloa/metabolismo , Ecótipo , Liases/genética , Liases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
18.
J Agric Food Chem ; 68(10): 3267-3276, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32101430

RESUMO

Cross-talk between various hormones is important in regulating many aspects of plant growth, development, and senescence, including fruit ripening. Here, exogenous ethylene (ETH, 100 µL/L, 12 h) rapidly accelerated 'Hayward' kiwifruit (Actinidia deliciosa) softening and ethylene production and was enhanced by supplementing with continuous treatment with methyl jasmonate (MeJA, 100 µM/L, 12 h) (ETH+MeJA). ETH+MeJA enhanced ACC synthase (ACS) activities and 1-aminocyclopropane-1-carboxylic acid (ACC) accumulation but not ACC oxidase (ACO) activity. Increased transcripts of ACS genes AdACS1 and AdACS2, ACS activity, and ethylene production were positively correlated. The abundance of AdACS1 was about 6-fold higher than AdACS2. RNA-seq identified 6 transcription factors among the 87 differentially expressed unigenes induced by ETH+MeJA. Dual-luciferase and electrophoretic mobility shift assays (EMSA) indicated that AdNAC2/3 physically interacted with and trans-activated the AdACS1 promoter 2.2- and 3.5-fold, respectively. Collectively, our results indicate that MeJA accelerates ethylene production in kiwifruit induced by exogenous ethylene, via a preferential activation of AdACS1 and AdACS2.


Assuntos
Acetatos/farmacologia , Actinidia/efeitos dos fármacos , Coenzima A Ligases/metabolismo , Ciclopentanos/farmacologia , Etilenos/biossíntese , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Oxilipinas/farmacologia , Reguladores de Crescimento de Planta/farmacologia , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Actinidia/enzimologia , Actinidia/genética , Actinidia/metabolismo , Frutas/efeitos dos fármacos , Frutas/enzimologia , Frutas/genética , Frutas/metabolismo , Proteínas de Plantas/genética , Fatores de Transcrição/genética
19.
Biochemistry ; 59(10): 1113-1123, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32101684

RESUMO

Steroid-degrading bacteria, including Mycobacterium tuberculosis (Mtb), utilize an architecturally distinct subfamily of acyl coenzyme A dehydrogenases (ACADs) for steroid catabolism. These ACADs are α2ß2 heterotetramers that are usually encoded by adjacent fadE-like genes. In mycobacteria, ipdE1 and ipdE2 (formerly fadE30 and fadE33) occur in divergently transcribed operons associated with the catabolism of 3aα-H-4α(3'-propanoate)-7aß-methylhexahydro-1,5-indanedione (HIP), a steroid metabolite. In Mycobacterium smegmatis, ΔipdE1 and ΔipdE2 mutants had similar phenotypes, showing impaired growth on cholesterol and accumulating 5-OH HIP in the culture supernatant. Bioinformatic analyses revealed that IpdE1 and IpdE2 share many of the features of the α- and ß-subunits, respectively, of heterotetrameric ACADs that are encoded by adjacent genes in many steroid-degrading proteobacteria. When coproduced in a rhodococcal strain, IpdE1 and IpdE2 of Mtb formed a complex that catalyzed the dehydrogenation of 5OH-HIP coenzyme A (5OH-HIP-CoA) to 5OH-3aα-H-4α(3'-prop-1-enoate)-7aß-methylhexa-hydro-1,5-indanedione coenzyme A ((E)-5OH-HIPE-CoA). This corresponds to the initial step in the pathway that leads to degradation of steroid C and D rings via ß-oxidation. Small-angle X-ray scattering revealed that the IpdE1-IpdE2 complex was an α2ß2 heterotetramer typical of other ACADs involved in steroid catabolism. These results provide insight into an important class of steroid catabolic enzymes and a potential virulence determinant in Mtb.


Assuntos
Acil-CoA Desidrogenase/metabolismo , Acil-CoA Desidrogenase/fisiologia , Acil Coenzima A/metabolismo , Proteínas de Bactérias/metabolismo , Colesterol/metabolismo , Coenzima A/metabolismo , Coenzima A Ligases/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Esteroides/metabolismo
20.
Arch Biochem Biophys ; 683: 108276, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-31978400

RESUMO

A Pseudomonas CoA ligase (BadA) biocatalyzed aroyl CoA thioesters used by a downstream N-benzoyltransferase (NDTNBT) in a cascade reaction made aroyl analogs of the anticancer drug paclitaxel. BadA kept the high-cost aroyl CoA substrates at saturation for the downstream NDTNBT by recycling CoA when it was added as the limiting reactant. A deacylated taxane substrate N-debenzoyl-2'-deoxypaclitaxel was converted to its benzoylated product at a higher yield, compared to the converted yield in assays in which the BadA ligase chemistry was omitted, and benzoyl CoA was added as a cosubstrate. The resulting benzoylated product 2'-deoxypaclitaxel was made at 196% over the theoretical yield of product that could be made from the CoA added at 50 µM, and the cosubstrates benzoic acid (100 µM), and N-debenzoyl-2'-deoxypaclitaxel (500 µM) added in excess. In addition, a 2-O-benzoyltransferase (mTBT) was incubated with BadA, aroyl acids, CoA, a 2-O-debenzoylated taxane substrate, and cofactors under the CoA-recycling conditions established for the NDTNBT/BadA cascade. The mTBT/BadA combination also made various 2-O-aroylated products that could potentially function as next-generation baccatin III compounds. These ligase/benzoyltransferase cascade reactions show the feasibility of recycling aroyl CoA thioesters in vitro to make bioactive acyl analogs of paclitaxel precursors.


Assuntos
Alcaloides/química , Antineoplásicos/química , Coenzima A Ligases/metabolismo , Paclitaxel/análogos & derivados , Taxoides/química , Ácido Benzoico/química , Biocatálise , Hidrocarbonetos Aromáticos com Pontes , Ácidos Carboxílicos/química , Catálise , Escherichia coli , Cinética , Paclitaxel/química , Rodopseudomonas/enzimologia , Especificidade por Substrato
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