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1.
Gene ; 741: 144559, 2020 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-32169630

RESUMO

The fungi in order Mortierellales are attractive producers for long-chain polyunsaturated fatty acids (PUFAs). Here, the genome sequencing and assembly of a novel strain of Mortierella sp. BCC40632 were done, yielding 65 contigs spanning of 49,964,116 total bases with predicted 12,149 protein-coding genes. We focused on the acetyl-CoA in relevant to its derived metabolic pathways for biosynthesis of macromolecules with biological functions, including PUFAs, eicosanoids and carotenoids. By comparative genome analysis between Mortierellales and Mucorales, the signature genetic characteristics of the arachidonic acid-producing strains, including Δ5-desaturase and GLELO-like elongase, were also identified in the strain BCC40632. Remarkably, this fungal strain contained only n-6 pathway of PUFA biosynthesis due to the absence of Δ15-desaturase or ω3-desaturase gene in contrast to other Mortierella species. Four putative enzyme sequences in the eicosanoid biosynthetic pathways were identified in the strain BCC40632 and others Mortierellale fungi, but were not detected in the Mucorales. Another unique metabolic trait of the Mortierellales was the inability in carotenoid synthesis as a result of the lack of phytoene synthase and phytoene desaturase genes. The findings provide a perspective in strain optimization for production of tailored-made products with industrial applications.


Assuntos
Acetilcoenzima A/biossíntese , Ácido Araquidônico/genética , Genoma Fúngico/genética , Mortierella/metabolismo , Acetilcoenzima A/genética , Ácido Araquidônico/biossíntese , Vias Biossintéticas/genética , Ácidos Graxos Dessaturases/genética , Elongases de Ácidos Graxos/genética , Ácidos Graxos Insaturados/genética , Ácidos Graxos Insaturados/metabolismo , Mortierella/genética , Mucorales/genética , Mucorales/metabolismo , Ácido gama-Linolênico/genética , Ácido gama-Linolênico/metabolismo
2.
Biochim Biophys Acta Gene Regul Mech ; 1862(10): 194436, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31682939

RESUMO

Histone H2A.Z plays an essential role in regulating transcriptional rates and memory. Interestingly, H2A.Z-bound nucleosomes are located in both transcriptionally active and inactive promotors, with no clear understanding of the mechanisms via which it differentially regulates transcription. We hypothesized that its functions are mediated through recruitment of regulatory proteins to promoters. Using rapid chromatin immunoprecipitation-mass spectrometry, we uncovered the association of H2A.Z-bound chromatin with the metabolic enzymes, oxoglutarate dehydrogenase (OGDH) and acetyl-CoA acyltransferase 2 (ACAA2). Recombinant green florescence fusion proteins, combined with mutations of predicted nuclear localization signals, confirmed their nuclear localization and chromatin binding. Conclusively, chromatin immunoprecipitation-deep sequencing, confirmed the predominant association of OGDH and ACAA2 with H2A.Z-occupied transcription start sites and enhancers, the former of which we confirmed is conserved in both mouse and human tissue. Furthermore, H2A.Z-deficient human HAP1 cells exhibited reduced chromatin-bound metabolic enzymes, accompanied with reduced posttranslational histone modifications, including acetylation and succinylation. Specifically, knockdown of OGDH diminished H4 succinylation. Thus, the data reveal that select metabolic enzymes are assembled at active, H2A.Z-occupied, promoters, for potential site-directed production of metabolic intermediates that are required for histone modifications.


Assuntos
Acetilcoenzima A/genética , Acetil-CoA C-Aciltransferase/genética , Histonas/genética , Complexo Cetoglutarato Desidrogenase/genética , Acetilação , Animais , Cromatina/genética , Código das Histonas/genética , Humanos , Camundongos , Proteínas do Tecido Nervoso/genética , Nucleossomos/genética , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional/genética , Fatores de Transcrição/genética , Sítio de Iniciação de Transcrição
3.
PLoS Biol ; 17(10): e3000461, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31600191

RESUMO

Dendritic spine development is crucial for the establishment of excitatory synaptic connectivity and functional neural circuits. Alterations in spine morphology and density have been associated with multiple neurological disorders. Autism candidate gene disconnected-interacting protein homolog 2 A (DIP2A) is known to be involved in acetylated coenzyme A (Ac-CoA) synthesis and is primarily expressed in the brain regions with abundant pyramidal neurons. However, the role of DIP2A in the brain remains largely unknown. In this study, we found that deletion of Dip2a in mice induced defects in spine morphogenesis along with thin postsynaptic density (PSD), and reduced synaptic transmission of pyramidal neurons. We further identified that DIP2A interacted with cortactin, an activity-dependent spine remodeling protein. The binding activity of DIP2A-PXXP motifs (P, proline; X, any residue) with the cortactin-Src homology 3 (SH3) domain was critical for maintaining the level of acetylated cortactin. Furthermore, Dip2a knockout (KO) mice exhibited autism-like behaviors, including excessive repetitive behaviors and defects in social novelty. Importantly, acetylation mimetic cortactin restored the impaired synaptic transmission and ameliorated repetitive behaviors in these mice. Altogether, our findings establish an initial link between DIP2A gene variations in autism spectrum disorder (ASD) and highlight the contribution of synaptic protein acetylation to synaptic processing.


Assuntos
Acetilcoenzima A/genética , Transtorno do Espectro Autista/genética , Cortactina/genética , Espinhas Dendríticas/metabolismo , Morfogênese/genética , Proteínas Nucleares/genética , Processamento de Proteína Pós-Traducional , Acetilcoenzima A/deficiência , Acetilação , Motivos de Aminoácidos , Animais , Animais Recém-Nascidos , Transtorno do Espectro Autista/metabolismo , Transtorno do Espectro Autista/fisiopatologia , Sítios de Ligação , Cortactina/metabolismo , Espinhas Dendríticas/ultraestrutura , Modelos Animais de Doenças , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Teste de Complementação Genética , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Densidade Pós-Sináptica/metabolismo , Densidade Pós-Sináptica/ultraestrutura , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Células Piramidais/metabolismo , Células Piramidais/ultraestrutura , Transmissão Sináptica
4.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(12): 158513, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31465888

RESUMO

The biosynthetic pathways for most lipophilic metabolites share several common principles. These substances are built almost exclusively from acetyl-CoA as the donor for the carbon scaffold and NADPH is required for the reductive steps during biosynthesis. Due to their hydrophobicity, the end products are sequestered into the same cellular compartment, the lipid droplet. In this review, we will summarize the efforts in the metabolic engineering of yeasts for the production of two major hydrophobic substance classes, fatty acid-based lipids and isoprenoids, with regard to these common aspects. We will compare and discuss the results of genetic engineering strategies to construct strains with enhanced synthesis of the precursor acetyl-CoA and with modified redox metabolism for improved NADPH supply. We will also discuss the role of the lipid droplet in the storage of the hydrophobic product and review the strategies to either optimize this organelle for higher capacity or to achieve excretion of the product into the medium.


Assuntos
Ácidos Graxos/genética , Hemiterpenos/genética , Engenharia Metabólica/métodos , Leveduras/genética , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Vias Biossintéticas , Butadienos/metabolismo , Ácidos Graxos/metabolismo , Hemiterpenos/metabolismo , Microbiologia Industrial/métodos , Metabolismo dos Lipídeos , NADP/genética , NADP/metabolismo , Leveduras/metabolismo
5.
Genes Dev ; 33(7-8): 388-402, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30808659

RESUMO

Tgif1 (thymine-guanine-interacting factor 1) and Tgif2 repress gene expression by binding directly to DNA or interacting with transforming growth factor (TGF) ß-responsive SMADs. Tgifs are essential for embryogenesis and may function in tumor progression. By analyzing both gain and loss of Tgif function in a well-established mouse model of intestinal cancer, we show that Tgifs promote adenoma growth in the context of mutant Apc (adenomatous polyposis coli). Despite the tumor-suppressive role of TGFß signaling, transcriptome profiling of colon tumors suggests minimal effect of Tgifs on the TGFß pathway. Instead, it appears that Tgifs, which are up-regulated in Apc mutant colon tumors, contribute to reprogramming metabolic gene expression. Integrating gene expression data from colon tumors with other gene expression and chromatin-binding data identifies a set of direct Tgif target genes encoding proteins involved in acetyl CoA and pyruvate metabolism. Analysis of both tumor and nontumor tissues indicates that these genes are targets of Tgif repression in multiple settings, suggesting that this is a core Tgif function. We propose that Tgifs play an important role in regulating basic energy metabolism in normal cells, and that this function of Tgifs is amplified in some cancers.


Assuntos
Acetilcoenzima A/genética , Adenoma , Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Homeodomínio/metabolismo , Neoplasias Intestinais , Proteínas Repressoras/metabolismo , Adenoma/genética , Adenoma/fisiopatologia , Polipose Adenomatosa do Colo/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Metabolismo Energético/genética , Células HCT116 , Humanos , Mucosa Intestinal/fisiopatologia , Neoplasias Intestinais/genética , Neoplasias Intestinais/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL
6.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(5): 654-661, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30731133

RESUMO

Previous studies have shown that the cardiolipin (CL)-deficient yeast mutant, crd1Δ, has decreased levels of acetyl-CoA and decreased activities of the TCA cycle enzymes aconitase and succinate dehydrogenase. These biochemical phenotypes are expected to lead to defective TCA cycle function. In this study, we report that signaling and anaplerotic metabolic pathways that supplement defects in the TCA cycle are essential in crd1Δ mutant cells. The crd1Δ mutant is synthetically lethal with mutants in the TCA cycle, retrograde (RTG) pathway, glyoxylate cycle, and pyruvate carboxylase 1. Glutamate levels were decreased, and the mutant exhibited glutamate auxotrophy. Glyoxylate cycle genes were up-regulated, and the levels of glyoxylate metabolites succinate and citrate were increased in crd1Δ. Import of acetyl-CoA from the cytosol into mitochondria is essential in crd1Δ, as deletion of the carnitine-acetylcarnitine translocase led to lethality in the CL mutant. ß-oxidation was functional in the mutant, and oleate supplementation rescued growth defects. These findings suggest that TCA cycle deficiency caused by the absence of CL necessitates activation of anaplerotic pathways to replenish acetyl-CoA and TCA cycle intermediates. Implications for Barth syndrome, a genetic disorder of CL metabolism, are discussed.


Assuntos
Cardiolipinas/genética , Ciclo do Ácido Cítrico , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Cardiolipinas/metabolismo , Deleção de Genes , Glioxilatos/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
Metab Eng ; 53: 1-13, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30684584

RESUMO

Medium-chain (C6-C10) chemicals are important components of fuels, commodities and fine chemicals. Numerous exciting achievements have proven reversed ß-oxidation cycle as a promising platform to synthesize these chemicals. However, under native central carbon metabolism, energetic and redox constraints limit the efficient operation of reversed ß-oxidation cycle. Current fermentative platform has to use different chemically and energetically inefficient ways for acetyl-CoA and NADH biosynthesis, respectively. The characteristics such as supplementation of additional acetate and formate or high ATP requirement makes this platform incompatible with large-scale production. Here, an artificial micro-aerobic metabolism for energy and carbon-efficient conversion of glycerol to MCFAs was constructed to present solutions towards these barriers. After evaluating numerous bacteria pathways under micro-aerobic conditions, one synthetic metabolic step enabling biosynthesis of acetyl-CoA and NADH simultaneously, without any energy cost and additional carbon requirement, and reducing loss of carbon to carbon dioxide-emitting reactions, was conceived and successfully constructed. The pyruvate dehydrogenase from Enterococcus faecalis was identified and biochemically characterized, demonstrating the most suitable characteristics. Furthermore, the carbon and energy metabolism in Escherichia coli was rewired by the clustered regularly interspaced short palindromic repeats interference system, inhibiting native fermentation pathways outcompeting this synthetic step. The present engineered strain exhibited a 15.7-fold increase in MCFA titer compared with that of the initial strain, and produced 15.67 g/L MCFAs from the biodiesel byproduct glycerol in 3-L bioreactor without exogenous feed of acetate or formate, representing the highest MCFA titer reported to date. This work demonstrates this artificial micro-aerobic metabolism has the potential to enable the cost-effective, large-scale production of fatty acids and other value-added reduced chemicals.


Assuntos
Metabolismo Energético , Escherichia coli , Ácidos Graxos/biossíntese , Engenharia Metabólica , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Reatores Biológicos , Enterococcus faecalis/enzimologia , Enterococcus faecalis/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Graxos/genética , Complexo Piruvato Desidrogenase/biossíntese , Complexo Piruvato Desidrogenase/genética
8.
ACS Synth Biol ; 7(11): 2686-2697, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30346720

RESUMO

Most of the current methods for controlling the formation rate of a key protein or enzyme in cell factories rely on the manipulation of target genes within the pathway. In this article, we present a novel synthetic system for post-translational regulation of protein levels, FENIX, which provides both independent control of the steady-state protein level and inducible accumulation of target proteins. The FENIX device is based on the constitutive, proteasome-dependent degradation of the target polypeptide by tagging with a short synthetic, hybrid NIa/SsrA amino acid sequence in the C-terminal domain. Protein production is triggered via addition of an orthogonal inducer ( i.e., 3-methylbenzoate) to the culture medium. The system was benchmarked in Escherichia coli by tagging two fluorescent proteins (GFP and mCherry), and further exploited to completely uncouple poly(3-hydroxybutyrate) (PHB) accumulation from bacterial growth. By tagging PhaA (3-ketoacyl-CoA thiolase, first step of the route), a dynamic metabolic switch at the acetyl-coenzyme A node was established in such a way that this metabolic precursor could be effectively redirected into PHB formation upon activation of the system. The engineered E. coli strain reached a very high specific rate of PHB accumulation (0.4 h-1) with a polymer content of ca. 72% (w/w) in glucose cultures in a growth-independent mode. Thus, FENIX enables dynamic control of metabolic fluxes in bacterial cell factories by establishing post-translational synthetic switches in the pathway of interest.


Assuntos
Escherichia coli/metabolismo , Hidroxibutiratos/metabolismo , Engenharia Metabólica/métodos , Poliésteres/metabolismo , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise
9.
Proc Natl Acad Sci U S A ; 115(40): E9499-E9506, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30228117

RESUMO

Acetyl-CoA synthetase 2 (ACSS2) is a conserved nucleocytosolic enzyme that converts acetate to acetyl-CoA. Adult mice lacking ACSS2 appear phenotypically normal but exhibit reduced tumor burdens in mouse models of liver cancer. The normal physiological functions of this alternate pathway of acetyl-CoA synthesis remain unclear, however. Here, we reveal that mice lacking ACSS2 exhibit a significant reduction in body weight and hepatic steatosis in a diet-induced obesity model. ACSS2 deficiency reduces dietary lipid absorption by the intestine and also perturbs repartitioning and utilization of triglycerides from adipose tissue to the liver due to lowered expression of lipid transporters and fatty acid oxidation genes. In this manner, ACSS2 promotes the systemic storage or metabolism of fat according to the fed or fasted state through the selective regulation of genes involved in lipid metabolism. Thus, targeting ACSS2 may offer a therapeutic benefit for the treatment of fatty liver disease.


Assuntos
Acetato-CoA Ligase/metabolismo , Tecido Adiposo/metabolismo , Fígado Gorduroso/metabolismo , Regulação da Expressão Gênica , Metabolismo dos Lipídeos , Fígado/metabolismo , Acetato-CoA Ligase/genética , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Tecido Adiposo/patologia , Animais , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Fígado/patologia , Camundongos , Camundongos Knockout
10.
J Agric Food Chem ; 66(37): 9667-9678, 2018 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-30036051

RESUMO

Cichoric acid (CA), a polyphenol component from Echinacea purpurea, exhibits preventive effects on liver lipid-metabolism disorders in obesity. This research aimed to determine the role of circadian rhythm signaling during the process of CA-attenuated lipid accumulation in hepatocytes. In the current study, CA treatments improved cell morphology changes and hepatic lipid levels, which were triggered by free fatty acids (2:1, oleate: palmitate) in a dose-dependent way. Besides, CA (200 µM) regulated the circadian rhythm expressions of clock genes and the relatively shallow daily oscillations. Moreover, silencing Bmal1 significantly blocked the p-Akt/Akt pathway to 80.1% ± 1.5% and the p-GSK3ß/GSK3ß pathway to 64.7% ± 2.8% ( p < 0.05). Furthermore, silencing Bmal1 elevated the expressions of FAS and ACC to 122.4% ± 5.6% and 114.9% ± 1.7% in protein levels ( p < 0.05) and to 166.5% ± 18.5% and 131.4% ± 5.5% in mRNA levels ( p < 0.05). Therefore, our results demonstrated that CA has a Bmal1 resistance to lipid accumulation by enhancing the Akt/GSK3ß signaling pathways and modulating the downstream expressions related to lipid metabolism, which indicated that CA might be useful as a natural and promising nonalcoholic fatty liver diseases (NAFLD) modulator.


Assuntos
Fatores de Transcrição ARNTL/metabolismo , Ácidos Cafeicos/farmacologia , Echinacea/química , Ácidos Graxos não Esterificados/metabolismo , Hepatócitos/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Extratos Vegetais/farmacologia , Succinatos/farmacologia , Fatores de Transcrição ARNTL/genética , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/genética , PPAR alfa/genética , PPAR alfa/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transdução de Sinais/efeitos dos fármacos
11.
Metab Eng ; 48: 175-183, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29883803

RESUMO

Methane, the primary component of natural gas, is the second most abundant greenhouse gas (GHG) and contributes significantly to climate change. The conversion of methane to industrial platform chemicals provides an attractive opportunity to decrease GHG emissions and utilize this inexpensive and abundantly available gas as a carbon feedstock. While technologies exist for chemical conversion of methane to liquid fuels, the technical complexity of these processes mandate high capital expenditure, large-scale commercial facilities to leverage economies of scale that cannot be efficiently scaled down. Alternatively, bioconversion technologies capable of efficient small-scale operation with high carbon and energy efficiency can enable deployment at remote methane resources inaccessible to current chemical technologies. Aerobic obligate methanotrophs, specifically Methylomicrobium buryatense 5GB1, have recently garnered increased research interest for development of such bio-technologies. In this study, we demonstrate production of C-4 carboxylic acids non-native to the host, specifically crotonic and butyric acids, from methane in an engineered M. buryatense 5GB1C by diversion of carbon flux through the acetyl-CoA node of central 'sugar' linked metabolic pathways using reverse ß-oxidation pathway genes. The synthesis of short chain carboxylic acids through the acetyl-CoA node demonstrates the potential for engineering M. buryatense 5GB1 as a platform for bioconversion of methane to a number of value added industrial chemicals, and presents new opportunities for further diversifying the products obtainable from methane as the feedstock.


Assuntos
Acetilcoenzima A , Ácido Butírico/metabolismo , Crotonatos/metabolismo , Engenharia Metabólica , Metano/metabolismo , Methylococcaceae , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Methylococcaceae/genética , Methylococcaceae/metabolismo
12.
Appl Environ Microbiol ; 84(15)2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29776932

RESUMO

Candida glabrata is a promising microorganism for the production of organic acids. Here, we report deletion and quantitative-expression approaches to elucidate the role of C. glabrata Med3AB (CgMed3AB), a subunit of the mediator transcriptional coactivator, in regulating cell growth. Deletion of CgMed3AB caused an 8.6% decrease in final biomass based on growth curve plots and 10.5% lower cell viability. Based on transcriptomics data, the reason for this growth defect was attributable to changes in expression of genes involved in pyruvate and acetyl-coenzyme A (CoA)-related metabolism in a Cgmed3abΔ strain. Furthermore, the mRNA level of acetyl-CoA synthetase was downregulated after deleting Cgmed3ab, resulting in 22.8% and 21% lower activity of acetyl-CoA synthetase and cellular acetyl-CoA, respectively. Additionally, the mRNA level of CgCln3, whose expression depends on acetyl-CoA, was 34% lower in this strain. As a consequence, the cell size and budding index in the Cgmed3abΔ strain were both reduced. Conversely, overexpression of Cgmed3ab led to 16.8% more acetyl-CoA and 120% higher CgCln3 mRNA levels, as well as 19.1% larger cell size and a 13.3% higher budding index than in wild-type cells. Taken together, these results suggest that CgMed3AB regulates cell growth in C. glabrata by coordinating homeostasis between cellular acetyl-CoA and CgCln3.IMPORTANCE This study demonstrates that CgMed3AB can regulate cell growth in C. glabrata by coordinating the homeostasis of cellular acetyl-CoA metabolism and the cell cycle cyclin CgCln3. Specifically, we report that CgMed3AB regulates the cellular acetyl-CoA level, which induces the transcription of Cgcln3, finally resulting in alterations to the cell size and budding index. In conclusion, we report that CgMed3AB functions as a wheel responsible for driving cellular acetyl-CoA metabolism, indirectly inducing the transcription of Cgcln3 and coordinating cell growth. We propose that Mediator subunits may represent a vital regulatory target modulating cell growth in C. glabrata.


Assuntos
Candida glabrata/citologia , Candida glabrata/metabolismo , Proteínas Fúngicas/metabolismo , Fatores de Transcrição/metabolismo , Acetato-CoA Ligase/genética , Acetato-CoA Ligase/metabolismo , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Candida glabrata/genética , Candida glabrata/crescimento & desenvolvimento , Ciclo Celular , Divisão Celular , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Esporos Fúngicos/citologia , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Fatores de Transcrição/genética
13.
Eur J Pharmacol ; 830: 76-86, 2018 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-29704496

RESUMO

Salusin-α is an endogenous bioactive peptide and likely to prevent atherosclerosis. But its protective effect against atherosclerosis in vivo remains poorly understood. The aim of the present study was to determine the potential effects of salusin-α on atherosclerosis and its associated metabolic disorders in high fat diet (HFD)-fed low density lipoprotein receptor deficient (LDLr-/-) mice, and also explore the possible underlying mechanisms involved. Our data showed that after 12 weeks treatment, salusin-α ameliorated HFD-induced weight gain, hyperlipidemia, and serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Salusin-α suppressed HFD-induced hepatic steatosis and regulated gene expression of fatty acid synthase, acetyl coenzyme A carboxylase-α, peroxisome proliferator-activated receptor-α, camitine palmitoyltransferase-1α and CYP7A1 in liver. Salusin-α reduced atherosclerotic plaque area and macrophage foam cell formation. Salusin-α prevented hepatic and aortic inflammation as evidenced by the reduced macrophage recruitment and mRNA expression of IL-6 and TNF-α in both liver and aorta. Salusin-α also reduced hepatic and aortic oxidative stress by normalizing activities of antioxidant enzymes in liver and suppressing reactive oxygen species generation and protein expressions of NADPH-oxidase (NOX) 2 and NOX4 in both liver and aorta. Our present data suggest that salusin-α could reduce hepatic steatosis and atherosclerosis via its pleiotropic effects, including amelioration of lipid profiles, regulation of some key molecules involved in lipid metabolism in liver, anti-oxidative effect and anti-inflammatory action.


Assuntos
Aterosclerose/tratamento farmacológico , Fígado Gorduroso/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Acetilcoenzima A/genética , Animais , Aterosclerose/sangue , Aterosclerose/genética , Carnitina O-Palmitoiltransferase/genética , Colesterol 7-alfa-Hidroxilase/genética , Dieta Hiperlipídica , Ácido Graxo Sintases/genética , Fígado Gorduroso/sangue , Fígado Gorduroso/genética , Hiperlipidemias/sangue , Hiperlipidemias/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Interleucina-6/sangue , Lipídeos/sangue , Masculino , Camundongos Knockout , PPAR alfa/genética , Receptores de LDL/genética , Fator de Necrose Tumoral alfa/sangue , Ganho de Peso/efeitos dos fármacos
14.
Curr Genet ; 64(4): 807-810, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29455333

RESUMO

Quiescent cells exploit an array of transcription factors to activate stress response machinery and maintain survival under nutrient-limited conditions. Our recent findings reveal that these transcription factors also play an important role in the exit of quiescence and regrowth. By studying Saccharomyces cerevisiae under a continuous, nutrient-limited condition, we found that Msn2 and Msn4 function as master regulators of glycolytic genes in the quiescent-like phase. They control the timing of transition from quiescence to growth by regulating the accumulation rate of acetyl-CoA, a key metabolite that is downstream of glycolysis and drives growth. These findings suggest a model that Msn2/4 not only protect the cells from starvation but also facilitate their regrowth from quiescence. Thus, understanding the functions of stress response transcription factors in metabolic regulation will provide deeper insight into how quiescent cells manage the capacity of regrowth.


Assuntos
Acetilcoenzima A/genética , Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Acetilcoenzima A/metabolismo , Glicólise/genética , Saccharomyces cerevisiae/metabolismo , Inanição/genética
15.
J Struct Biol ; 202(1): 70-81, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29241954

RESUMO

In bacteria, biosynthesis of riboflavin occurs through a series of enzymatic steps starting with one molecule of GTP and two molecules of ribulose-5-phosphate. In Bacillus subtilis (B. subtilis) the genes (ribD/G, ribE, ribA, ribH and ribT) which are involved in riboflavin biosynthesis are organized in an operon referred as rib operon. All the genes of rib operon are characterized functionally except for ribT. The ribT gene with unknown function is found at the distal terminal of rib operon and annotated as a putative N-acetyltransferase. Here, we report the crystal structure of ribT from B. subtilis (bribT) complexed with coenzyme A (CoA) at 2.1 Šresolution determined by single wavelength anomalous dispersion method. Our structural study reveals that bribT is a member of GCN5-related N-acetyltransferase (GNAT) superfamily and contains all the four conserved structural motifs that have been in other members of GNAT superfamily. The members of GNAT family transfers the acetyl group from acetyl coenzyme A (AcCoA) to a variety of substrates. Moreover, the structural analysis reveals that the residues Glu-67 and Ser-107 are suitably positioned to act as a catalytic base and catalytic acid respectively suggesting that the catalysis by bribT may follow a direct transfer mechanism. Surprisingly, the mutation of a non-conserved amino acid residue Cys-112 to alanine or serine affected the binding of AcCoA to bribT, indicating a possible role of Cys-112 in the catalysis.


Assuntos
Acetiltransferases/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Óperon , Acetilcoenzima A/química , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Acetiltransferases/química , Acetiltransferases/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Domínio Catalítico/genética , Cristalografia por Raios X , Modelos Moleculares , Mutação , Ligação Proteica , Riboflavina/biossíntese
16.
ACS Synth Biol ; 7(1): 24-29, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-28945971

RESUMO

Efficient utilization of lignocellulose is pivotal for economically converting renewable feedstocks into value-added products. Xylose is the second most abundant sugar in lignocellulose, but it is quite challenging to ferment xylose as efficiently as glucose by microorganisms. Here, we investigated the metabolic potential of three xylose catabolic pathways (isomerase, Weimberg, and Dahms pathways) and illustrated the synergetic effect between the isomerase pathway and Weimberg pathway for the synthesis of chemicals derived from 2-ketoglutarate and acetyl-CoA. When using glutaric acid as the target product, employment of such synergetic pathways in combination resulted in an increased glutaric acid titer (602 mg/L) compared with using each pathway alone (104 or 209 mg/L), and this titer even outcompetes that obtained from the glucose catabolic pathway for glutaric acid synthesis (420 mg/L). This work validates a novel and powerful strategy for xylose metabolic utilization to overcome the inefficiency of using a single xylose metabolic pathway for the synthesis of TCA cycle derived chemicals.


Assuntos
Glutaratos/metabolismo , Engenharia Metabólica , Xilose/metabolismo , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Ciclo do Ácido Cítrico , Escherichia coli/metabolismo , Glutaratos/análise , Isomerases/genética , Isomerases/metabolismo , Ácidos Cetoglutáricos/metabolismo , Lignina/química , Lignina/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Espectrofotometria Ultravioleta
17.
Metab Eng ; 44: 313-324, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-29122703

RESUMO

Previous studies have made many exciting achievements on pushing the functional reversal of beta-oxidation cycle (r-BOX) to more widespread adoption for synthesis of a wide variety of fuels and chemicals. However, the redox cofactor requirement for the efficient operation of r-BOX remains unclear. In this work, the metabolic efficiency of r-BOX for medium-chain fatty acid (C6-C10, MCFA) production was optimized by redox cofactor engineering. Stoichiometric analysis of the r-BOX pathway and further experimental examination identified NADH as a crucial determinant of r-BOX process yield. Furthermore, the introduction of formate dehydrogenase from Candida boidinii using fermentative inhibitor byproduct formate as a redox NADH sink improved MCFA titer from initial 1.2g/L to 3.1g/L. Moreover, coupling of increasing the supply of acetyl-CoA with NADH to achieve fermentative redox balance enabled product synthesis at maximum titers. To this end, the acetate re-assimilation pathway was further optimized to increase acetyl-CoA availability associated with the new supply of NADH. It was found that the acetyl-CoA synthetase activity and intracellular ATP levels constrained the activity of acetate re-assimilation pathway, and 4.7g/L of MCFA titer was finally achieved after alleviating these two limiting factors. To the best of our knowledge, this represented the highest titer reported to date. These results demonstrated that the key constraint of r-BOX was redox imbalance and redox engineering could further unleash the lipogenic potential of this cycle. The redox engineering strategies could be applied to acetyl-CoA-derived products or other bio-products requiring multiple redox cofactors for biosynthesis.


Assuntos
Acetilcoenzima A , Candida , Coenzimas , Ácidos Graxos , NADP , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Candida/genética , Candida/metabolismo , Coenzimas/genética , Coenzimas/metabolismo , Ácidos Graxos/biossíntese , Ácidos Graxos/genética , NADP/genética , NADP/metabolismo , Oxirredução
18.
Sci Rep ; 7(1): 14790, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-29093482

RESUMO

Reversible Nε-lysine acetylation has emerging as an important metabolic regulatory mechanism in microorganisms. Herein, we systematically investigated the site-specific and kinetic characterization of enzymatic (lysine acetyltransferase) and nonenzymatic acetylation (AcP-dependent or Acyl-CoA-dependent), as well as their different effect on activity of metabolic enzyme (AMP-forming acetyl-CoA synthetase, Acs). It was found that Bacillus subtilis acetyl-CoA synthetase (BsAcsA) can be acetylated in vitro either catalytically by lysine acetyltransferase BsAcuA and Ac-CoA (at low concentration), or nonenzymatically by Ac-CoA or AcP (at high concentration). Two distinct mechanisms show preference for different lysine acetylation site (enzymatic acetylation for K549 and nonenzymatic acetylation for K524), and reveal different dynamics of relative acetylation changes at these lysine sites. The results demonstrated that lysine residues on the same protein exhibit different acetylation reactivity with acetyl-phosphate and acetyl-CoA, which was determined by surface accessibility, three-dimensional microenvironment, and pKa value of lysine. Acetyl-CoA synthetase is inactivated by AcuA-catalyzed acetylation, but not by nonenzymatic acetylation.


Assuntos
Acetato-CoA Ligase/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lisina Acetiltransferases/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Acetato-CoA Ligase/genética , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Acetilação , Bacillus subtilis/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Lisina Acetiltransferases/genética
19.
Sci Rep ; 7(1): 12653, 2017 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-28978999

RESUMO

This study aimed to establish a therapeutic strategy targeting hypoxic cancer cells in gastric carcinoma (GC). YC-1 is a HIF-1α inhibitor, and we revealed that low-dose YC-1 (10 µM) suppressed HIF-1α expression, and induced hypoxia-dependent apoptosis in the GC cell line 58As9. This hypoxia-specific apoptosis induction by YC-1 involved excessive reactive oxygen species (ROS) generation. The apoptotic effect of 10 µM YC-1 was enhanced by additional glucose (G) and insulin (I) treatments. RT-PCR demonstrated that 10 µM YC-1 reduced hypoxia-induced expression of HIF-1α targets involved in anaerobic glycolysis. Metabolic analysis showed that YC-1 shifted glucose metabolism in hypoxic cells from anaerobic glycolysis to oxidative phosphorylation (OXPHOS). Additional GI accelerated membranous GLUT1 translocation, elevating glucose uptake, and increased acetyl-CoA levels, leading to more ROS generation in hypoxic YC-1-treated cells. Finally, we evaluated the anti-cancer effect of low-dose YC-1 (1 mg/kg) + G (2 g/kg) and I (1 unit/3 g G) treatment in xenograft models. YC-1 + GI therapy strongly inhibited tumour growth. Immunohistochemical analysis demonstrated that YC-1 + GI reduced HIF-1α expression and pimonidazole accumulation in tumours. Conversely, YC-1 + GI increased intra-tumoral 8-OHdG and levels of apoptosis markers. Low-dose YC-1 + GI is a unique therapy targeting hypoxic GC cells that generates lethal ROS via forced activation of OXPHOS.


Assuntos
Carcinoma/tratamento farmacológico , Transportador de Glucose Tipo 1/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Indazóis/administração & dosagem , Neoplasias Gástricas/tratamento farmacológico , Acetilcoenzima A/genética , Anaerobiose/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Desoxiguanosina/administração & dosagem , Desoxiguanosina/análogos & derivados , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Insulina/metabolismo , Camundongos , Nitroimidazóis/administração & dosagem , Fosforilação Oxidativa/efeitos dos fármacos , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Hipóxia Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Metab Eng ; 42: 126-133, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28627452

RESUMO

Yarrowia lipolytica is considered as a potential candidate for succinic acid production because of its innate ability to accumulate citric acid cycle intermediates and its tolerance to acidic pH. Previously, a succinate-production strain was obtained through the deletion of succinate dehydrogenase subunit encoding gene Ylsdh5. However, the accumulation of by-product acetate limited further improvement of succinate production. Meanwhile, additional pH adjustment procedure increased the downstream cost in industrial application. In this study, we identified for the first time that acetic acid overflow is caused by CoA-transfer reaction from acetyl-CoA to succinate in mitochondria rather than pyruvate decarboxylation reaction in SDH negative Y. lipolytica. The deletion of CoA-transferase gene Ylach eliminated acetic acid formation and improved succinic acid production and the cell growth. We then analyzed the effect of overexpressing the key enzymes of oxidative TCA, reductive carboxylation and glyoxylate bypass on succinic acid yield and by-products formation. The best strain with phosphoenolpyruvate carboxykinase (ScPCK) from Saccharomyces cerevisiae and endogenous succinyl-CoA synthase beta subunit (YlSCS2) overexpression improved succinic acid titer by 4.3-fold. In fed-batch fermentation, this strain produced 110.7g/L succinic acid with a yield of 0.53g/g glycerol without pH control. This is the highest succinic acid titer achieved at low pH by yeast reported worldwide, to date, using defined media. This study not only revealed the mechanism of acetic acid overflow in SDH negative Y. lipolytica, but it also reported the development of an efficient succinic acid production strain with great industrial prospects.


Assuntos
Glicerol/metabolismo , Engenharia Metabólica , Ácido Succínico/metabolismo , Yarrowia/metabolismo , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Ciclo do Ácido Cítrico/genética , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Concentração de Íons de Hidrogênio , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , Yarrowia/genética
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