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1.
FASEB J ; 36(1): e22078, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34918381

RESUMO

Large clinical trials and real-world studies have demonstrated that the beneficial effects of sodium-glucose co-transporter 2 (SGLT2) inhibitors on renal outcomes regardless of the presence of diabetes. However, the mechanism remains obscure. Here, we analyze the anti-fibrotic and anti-inflammatory effects of dapagliflozin, a SGLT2 inhibitor, on renal alternations using the ischemia/reperfusion-induced fibrosis model. Transcriptome and metabolome analysis showed that the accumulation of tricarboxylic acid (TCA) cycle metabolites and upregulation of inflammation in fibrosis renal cortical tissue were mitigated by dapagliflozin treatment. Moreover, dapagliflozin markedly relieved the activation of mammalian target of rapamycin and hypoxia inducible factor-1α signaling and restored tubular cell-preferred fatty acid oxidation. Notably, NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation was strikingly blocked by dapagliflozin. We further demonstrated that the immunomodulatory metabolite itaconate derived from the TCA cycle was significantly boosted as a result of decreased isocitrate dehydrogenase 2 and increased immune-responsive gene 1 and mitochondrial citrate carrier in dapagliflozin-treated mice, which contributed to the inhibitory effect of dapagliflozin on NLRP3 inflammasome activation. Furthermore, administration of cell-permeable itaconate surrogate prevented activation of NLRP3 inflammasome and protected kidney against fibrosis development. Our results identify a novel mechanism coupling metabolism and inflammation for kidney benefits of SGLT2 inhibition in progressive chronic kidney disease.


Assuntos
Compostos Benzidrílicos/farmacologia , Glucosídeos/farmacologia , Inflamassomos/metabolismo , Nefropatias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Transportador 2 de Glucose-Sódio/metabolismo , Succinatos/metabolismo , Animais , Ciclo do Ácido Cítrico/efeitos dos fármacos , Fibrose , Nefropatias/tratamento farmacológico , Masculino , Camundongos
2.
FASEB J ; 35(10): e21936, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34547129

RESUMO

Lipopolysaccharide (LPS)-stimulated macrophages express an aconitate decarboxylase (IRG1, also called ACOD1), leading to accumulation of the endogenous metabolite itaconate. However, the precise mechanisms by which elevated itaconate levels alter macrophage function are not clear. Our hypothesis is itaconate affects macrophage function through some uncertain mechanism. Based on this, we established a transcriptional and proteomic signature of macrophages stimulated by itaconate and identified the pathways of IL-1ß secretion and altered iron metabolism. Consistently, the effect of IRG1 deficiency on IL-1ß secretion and iron metabolism was confirmed in IRG1 knockout THP-1 cell lines. Several common inhibitors and other compounds were used to examine the molecular mechanisms involved. Only cysteine and antioxidants (catechin hydrate) could inhibit caspase-1 activation and IL-1ß secretion in itaconate-stimulated macrophages. We further found that aconitase activity was decreased by itaconate stimulation. Our results demonstrate the counteracting effects of overexpression of mitochondrial aconitase (ACO2, a tricarboxylic acid cycle enzyme) or cytosolic aconitase (ACO1, an iron regulatory protein) on IL-1ß secretion and altered iron metabolism. Both enzyme activities were inhibited by itaconate because of iron-sulfur (Fe-S) cluster destruction. Our findings indicate that the immunoregulatory functions of IRG1 and itaconate in macrophages are stressful Fe-S cluster of aconitases disrupting and iron metabolism rebalancing.


Assuntos
Ferro/metabolismo , Macrófagos/metabolismo , Succinatos/metabolismo , Enxofre/metabolismo , Aconitato Hidratase/metabolismo , Carboxiliases/metabolismo , Linhagem Celular , Cisteína/metabolismo , Citocinas/metabolismo , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteoma/metabolismo , Proteômica
3.
Biomolecules ; 11(8)2021 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-34439834

RESUMO

The glutarylation of lysine residues in proteins attracts attention as a possible mechanism of metabolic regulation, perturbed in pathologies. The visualization of protein glutarylation by antibodies specific to ε-glutaryl-lysine residues may be particularly useful to reveal pathogenic mutations in the relevant enzymes. We purified such antibodies from the rabbit antiserum, obtained after sequential immunization with two artificially glutarylated proteins, using affinity chromatography on ε-glutaryl-lysine-containing sorbents. Employing these anti(ε-glutaryl-lysine)-antibodies for the immunoblotting analysis of rat tissues and mitochondria has demonstrated the sample-specific patterns of protein glutarylation. The study of the protein glutarylation in rat tissue homogenates revealed a time-dependent fragmentation of glutarylated proteins in these preparations. The process may complicate the investigation of potential changes in the acylation level of specific protein bands when studying time-dependent effects of the acylation regulators. In the rat brain, the protein glutarylation, succinylation and acetylation patterns obtained upon the immunoblotting of the same sample with the corresponding antibodies are shown to differ. Specific combinations of molecular masses of major protein bands in the different acylation patterns confirm the selectivity of the anti(ε-glutaryl-lysine)-antibodies obtained in this work. Hence, our affinity-purified anti(ε-glutaryllysine)-antibodies provide an effective tool to characterize protein glutarylation, revealing its specific pattern, compared to acetylation and succinylation, in complex protein mixtures.


Assuntos
Glutaratos/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Succinatos/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos/isolamento & purificação , Especificidade de Anticorpos , Encéfalo/metabolismo , Cromatografia de Afinidade , Soros Imunes/química , Immunoblotting , Fígado/metabolismo , Masculino , Coelhos , Ratos
4.
Int J Mol Sci ; 22(16)2021 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-34445761

RESUMO

Natural products of microbial origin have inspired most of the commercial pharmaceuticals, especially those from Actinobacteria. However, the redundancy of molecules in the discovery process represents a serious issue. The untargeted approach, One Strain Many Compounds (OSMAC), is one of the most promising strategies to induce the expression of silent genes, especially when combined with genome mining and advanced metabolomics analysis. In this work, the whole genome of the marine isolate Rhodococcus sp. I2R was sequenced and analyzed by antiSMASH for the identification of biosynthetic gene clusters. The strain was cultivated in 22 different growth media and the generated extracts were subjected to metabolomic analysis and functional screening. Notably, only a single growth condition induced the production of unique compounds, which were partially purified and structurally characterized by liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). This strategy led to identifying a bioactive fraction containing >30 new glycolipids holding unusual functional groups. The active fraction showed a potent antiviral effect against enveloped viruses, such as herpes simplex virus and human coronaviruses, and high antiproliferative activity in PC3 prostate cancer cell line. The identified compounds belong to the biosurfactants class, amphiphilic molecules, which play a crucial role in the biotech and biomedical industry.


Assuntos
Antivirais/metabolismo , Glicolipídeos/metabolismo , Rhodococcus/metabolismo , Animais , Antivirais/análise , Chlorocebus aethiops , Técnicas de Cultura , Ensaios de Seleção de Medicamentos Antitumorais , Ésteres/metabolismo , Genoma Bacteriano , Glicolipídeos/química , Humanos , Metaboloma , Testes de Sensibilidade Microbiana , Estrutura Molecular , Células PC-3 , Rhodococcus/química , Rhodococcus/genética , Succinatos/metabolismo , Tensoativos/química , Tensoativos/metabolismo , Células Vero
5.
Infect Immun ; 89(10): e0015621, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34125603

RESUMO

Brucellosis is one of the most common global zoonoses and is caused by facultative intracellular bacteria of the genus Brucella. Numerous studies have found that MyD88 signaling contributes to protection against Brucella; however, the underlying mechanism has not been entirely defined. Here, we show that MyD88 signaling in hematopoietic cells contributes both to inflammation and to control of Brucella melitensis infection in vivo. While the protective role of MyD88 in Brucella infection has often been attributed to promotion of gamma interferon (IFN-γ) production, we found that MyD88 signaling restricts host colonization by B. melitensis even in the absence of IFN-γ. In vitro, we show that MyD88 promotes macrophage glycolysis in response to B. melitensis. Interestingly, a B. melitensis mutant lacking the glucose transporter, GluP, was more highly attenuated in MyD88-/- than in wild-type mice, suggesting MyD88 deficiency results in an increased availability of glucose in vivo, which Brucella can exploit via GluP. Metabolite profiling of macrophages identified several metabolites regulated by MyD88 in response to B. melitensis, including itaconate. Subsequently, we found that itaconate has antibacterial effects against Brucella and also regulates the production of proinflammatory cytokines in B. melitensis-infected macrophages. Mice lacking the ability to produce itaconate were also more susceptible to B. melitensis in vivo. Collectively, our findings indicate that MyD88-dependent changes in host metabolism contribute to control of Brucella infection.


Assuntos
Brucelose/metabolismo , Glucose/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Succinatos/metabolismo , Animais , Brucella melitensis/patogenicidade , Brucelose/microbiologia , Citocinas/metabolismo , Glicólise/fisiologia , Interferon gama/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia
6.
PLoS One ; 16(5): e0251067, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33951112

RESUMO

During the glyoxylate cycle, isocitrate lyases (ICLs) catalyze the lysis of isocitrate to glyoxylate and succinate. Itaconate has been reported to inhibit an ICL from Mycobacterium tuberculosis (tbICL). To elucidate the molecular mechanism of ICL inhibition, we determined the crystal structure of tbICL in complex with itaconate. Unexpectedly, succinate and itaconate were found to bind to the respective active sites in the dimeric form of tbICL. Our structure revealed the active site architecture as an open form, although the substrate and inhibitor were bound to the active sites. Our findings provide novel insights into the conformation of tbICL upon its binding to a substrate or inhibitor, along with molecular details of the inhibitory mechanism of itaconate.


Assuntos
Isocitrato Liase/química , Isocitrato Liase/metabolismo , Isocitratos/química , Succinatos/química , Succinatos/metabolismo , Ácido Succínico/química , Ácido Succínico/metabolismo , Catálise , Domínio Catalítico/fisiologia , Glioxilatos/química , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Conformação Proteica
7.
J Neurochem ; 158(2): 262-281, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33837559

RESUMO

Tissue accumulation and high urinary excretion of ethylmalonic acid (EMA) are found in ethylmalonic encephalopathy (EE), an inherited disorder associated with cerebral and cerebellar atrophy whose pathogenesis is poorly established. The in vitro and in vivo effects of EMA on bioenergetics and redox homeostasis were investigated in rat cerebellum. For the in vitro studies, cerebellum preparations were exposed to EMA, whereas intracerebellar injection of EMA was used for the in vivo evaluation. EMA reduced state 3 and uncoupled respiration in vitro in succinate-, glutamate-, and malate-supported mitochondria, whereas decreased state 4 respiration was observed using glutamate and malate. Furthermore, mitochondria permeabilization and succinate supplementation diminished the decrease in state 3 with succinate. EMA also inhibited the activity of KGDH, an enzyme necessary for glutamate oxidation, in a mixed manner and augmented mitochondrial efflux of α-ketoglutarate. ATP levels were markedly reduced by EMA, reflecting a severe bioenergetic disruption. Docking simulations also indicated interactions between EMA and KGDH and a competition with glutamate and succinate for their mitochondrial transporters. In vitro findings also showed that EMA decreased mitochondrial membrane potential and Ca2+ retention capacity, and induced swelling in the presence of Ca2+ , which were prevented by cyclosporine A and ADP and ruthenium red, indicating mitochondrial permeability transition (MPT). Moreover, EMA, at high concentrations, mildly increased ROS levels and altered antioxidant defenses in vitro and in vivo. Our data indicate that EMA-induced impairment of glutamate and succinate oxidation and MPT may contribute to the pathogenesis of the cerebellum abnormalities in EE.


Assuntos
Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Metabolismo Energético/efeitos dos fármacos , Glutamatos/metabolismo , Malonatos/toxicidade , Poro de Transição de Permeabilidade Mitocondrial , Succinatos/metabolismo , Animais , Ácidos Cetoglutáricos/metabolismo , Malatos/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Simulação de Acoplamento Molecular , Oxirredução , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos Wistar , Succinatos/farmacologia
8.
Nat Commun ; 12(1): 2261, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859194

RESUMO

Expanding the portfolio of products that can be made from lignin will be critical to enabling a viable bio-based economy. Here, we engineer Pseudomonas putida for high-yield production of the tricarboxylic acid cycle-derived building block chemical, itaconic acid, from model aromatic compounds and aromatics derived from lignin. We develop a nitrogen starvation-detecting biosensor for dynamic two-stage bioproduction in which itaconic acid is produced during a non-growth associated production phase. Through the use of two distinct itaconic acid production pathways, the tuning of TCA cycle gene expression, deletion of competing pathways, and dynamic regulation, we achieve an overall maximum yield of 56% (mol/mol) and titer of 1.3 g/L from p-coumarate, and 1.4 g/L titer from monomeric aromatic compounds produced from alkali-treated lignin. This work illustrates a proof-of-principle that using dynamic metabolic control to reroute carbon after it enters central metabolism enables production of valuable chemicals from lignin at high yields by relieving the burden of constitutively expressing toxic heterologous pathways.


Assuntos
Lignina/metabolismo , Engenharia Metabólica/métodos , Pseudomonas putida/metabolismo , Succinatos/metabolismo , Álcalis/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Basidiomycota/enzimologia , Basidiomycota/genética , Técnicas Biossensoriais , Burkholderia/enzimologia , Burkholderia/genética , Carbono/metabolismo , Ciclo do Ácido Cítrico/genética , Ácidos Cumáricos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Microbiologia Industrial/métodos , Lignina/química , Estudo de Prova de Conceito , Pseudomonas putida/genética
9.
Anal Bioanal Chem ; 413(9): 2493-2501, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33665672

RESUMO

Sample preparation methodology is critical to obtaining reliable data for studying endogenous metabolites. Dependable preparation techniques require separation of cells from culture media, quenching of enzymatic activity, and extraction of metabolites from the cells. Presented here is a simple, rapid, semi-automated metabolomic sample preparation technique for 20 µL samples of RAW 264.7 cells suspended in culture media. This method uses online filter-assisted electroporation-based cell lysis and chilled organic solvent extraction to prepare metabolomic samples from cells in suspension in 2 min. Experiments using an isotopically labeled adenosine triphosphate internal standard were carried out to ensure enzymatic quenching by monitoring the ratio of labeled adenosine diphosphate to adenosine triphosphate. Cells were metabolically labeled with 13C-glucose concurrent with sampling aliquots of the cell suspension over the course of 24 h. Incorporation of 13C into organic acid metabolites such as itaconate Cell lysates was analyzed by nano-reverse-phase liquid chromatography-mass spectrometry (nano-RP-LC-MS), showing incorporation of 13C into organic acid metabolites such as itaconate.


Assuntos
Metabolômica/métodos , Animais , Cromatografia Líquida , Glucose/metabolismo , Espectrometria de Massas , Metaboloma , Camundongos , Células RAW 264.7 , Succinatos/metabolismo
10.
Nat Commun ; 12(1): 1399, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33658521

RESUMO

Staphylococcus aureus is a prominent human pathogen that readily adapts to host immune defenses. Here, we show that, in contrast to Gram-negative pathogens, S. aureus induces a distinct airway immunometabolic response dominated by the release of the electrophilic metabolite, itaconate. The itaconate synthetic enzyme, IRG1, is activated by host mitochondrial stress, which is induced by staphylococcal glycolysis. Itaconate inhibits S. aureus glycolysis and selects for strains that re-direct carbon flux to fuel extracellular polysaccharide (EPS) synthesis and biofilm formation. Itaconate-adapted strains, as illustrated by S. aureus isolates from chronic airway infection, exhibit decreased glycolytic activity, high EPS production, and proficient biofilm formation even before itaconate stimulation. S. aureus thus adapts to the itaconate-dominated immunometabolic response by producing biofilms, which are associated with chronic infection of the human airway.


Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/fisiologia , Staphylococcus aureus/patogenicidade , Succinatos/metabolismo , Adulto , Animais , Biofilmes/crescimento & desenvolvimento , Líquido da Lavagem Broncoalveolar , Metabolismo dos Carboidratos , Fibrose Cística/microbiologia , Regulação Bacteriana da Expressão Gênica , Glicólise/efeitos dos fármacos , Glicólise/fisiologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Hidroliases/metabolismo , Camundongos Endogâmicos C57BL , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Escarro/microbiologia , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificação , Estresse Fisiológico , Succinatos/farmacologia , Ácido Succínico/metabolismo , Adulto Jovem
11.
Cancer Res ; 81(11): 2861-2873, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33762357

RESUMO

Defective mitosis with chromosome missegregation can have a dramatic effect on genome integrity by causing DNA damage, activation of the DNA damage response (DDR), and chromosomal instability. Although this is an energy-dependent process, mechanisms linking DDR to cellular metabolism are unknown. Here we show that checkpoint kinase 2 (CHK2), a central effector of DDR, regulates cellular energy production by affecting glycolysis and mitochondrial functions. Patients with hepatocellular carcinoma (HCC) had increased CHK2 mRNA in blood, which was associated with elevated tricarboxylic acid cycle (TCA) metabolites. CHK2 controlled expression of succinate dehydrogenase (SDH) and intervened with mitochondrial functions. DNA damage and CHK2 promoted SDH activity marked by increased succinate oxidation through the TCA cycle; this was confirmed in a transgenic model of HCC with elevated DNA damage. Mitochondrial analysis identified CHK2-controlled expression of SDH as key in sustaining reactive oxygen species production. Cells with DNA damage and elevated CHK2 relied significantly on glycolysis for ATP production due to dysfunctional mitochondria, which was abolished by CHK2 knockdown. This represents a vulnerability created by the DNA damage response that could be exploited for development of new therapies. SIGNIFICANCE: This study uncovers a link between a central effector of DNA damage response, CHK2, and cellular metabolism, revealing potential therapeutic strategies for targeting hepatocellular carcinoma.


Assuntos
Carcinoma Hepatocelular/patologia , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA , Glicólise , Neoplasias Hepáticas/patologia , Metaboloma , Transcriptoma , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Quinase do Ponto de Checagem 2/genética , Ciclo do Ácido Cítrico , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitose , Espécies Reativas de Oxigênio/metabolismo , Succinatos/metabolismo
12.
ACS Chem Biol ; 16(3): 463-470, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33688722

RESUMO

The isocitrate lyases (ICL1/2) are essential enzymes of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. At present, no ICL1/2 inhibitors have progressed to clinical evaluation, despite extensive drug discovery efforts. Herein, we surveyed succinate analogs against ICL1 and found that dicarboxylic acids constrained in their synperiplanar conformations, such as maleic acid, comprise uncompetitive inhibitors of ICL1 and inhibit more potently than their trans-isomers. From this, we identified cis-2,3 epoxysuccinic acid (cis-EpS) as a selective, irreversible covalent inactivator of Mtb ICL1 (kinact/Kinact= (5.0 ± 1.4) × 104 M-1 s-1; Kinact = 200 ± 50 nM), the most potent inactivator of ICL1 yet characterized. Crystallographic and mass spectrometric analysis demonstrated that Cys191 of ICL1 was S-malylated by cis-EpS, and a crystallographic "snapshot" of inactivation lent insight into the chemical mechanism of this inactivation. Proteomic analysis of E. coli lysates showed that cis-EpS selectively labeled plasmid-expressed Mtb ICL1. Consistently, cis-EpS, but not its trans-isomer, inhibited the growth of Mtb under conditions in which ICL function is essential. These findings encourage the development of analogs of cis-2,3-epoxysuccinate as antituberculosis agents.


Assuntos
Antituberculosos/química , Inibidores Enzimáticos/química , Isocitrato Liase/antagonistas & inibidores , Mycobacterium tuberculosis/enzimologia , Succinatos/química , Tuberculose/tratamento farmacológico , Antituberculosos/metabolismo , Descoberta de Drogas , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Escherichia coli/metabolismo , Glicolatos/química , Glioxilatos/química , Humanos , Isomerismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteômica , Succinatos/metabolismo , Termodinâmica
13.
Nat Commun ; 12(1): 1563, 2021 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-33692355

RESUMO

Purple coneflower (Echinacea purpurea (L.) Moench) is a popular native North American herbal plant. Its major bioactive compound, chicoric acid, is reported to have various potential physiological functions, but little is known about its biosynthesis. Here, taking an activity-guided approach, we identify two cytosolic BAHD acyltransferases that form two intermediates, caftaric acid and chlorogenic acid. Surprisingly, a unique serine carboxypeptidase-like acyltransferase uses chlorogenic acid as its acyl donor and caftaric acid as its acyl acceptor to produce chicoric acid in vacuoles, which has evolved its acyl donor specificity from the better-known 1-O-ß-D-glucose esters typical for this specific type of acyltransferase to chlorogenic acid. This unusual pathway seems unique to Echinacea species suggesting convergent evolution of chicoric acid biosynthesis. Using these identified acyltransferases, we have reconstituted chicoric acid biosynthesis in tobacco. Our results emphasize the flexibility of acyltransferases and their roles in the evolution of specialized metabolism in plants.


Assuntos
Aciltransferases/metabolismo , Ácidos Cafeicos/metabolismo , Echinacea/enzimologia , Echinacea/metabolismo , Proteínas de Plantas/metabolismo , Succinatos/metabolismo
14.
Trends Biotechnol ; 39(4): 330-331, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33632542

RESUMO

Due to low carboxylase activity, CO2 biotransformation is challenging to achieve using natural CO2 fixation pathways. Liu et al. have improved the activity of propionyl-CoA carboxylase (PCC) 94-fold, enabling the efficient synthesis of succinate from acetyl-CoA and paving the way for CO2 assimilation via the 3-hydroxypropionate (3-HP) bicycle or 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle.


Assuntos
Biotecnologia , Evolução Molecular Direcionada , Ácido Succínico , Biotecnologia/métodos , Dióxido de Carbono/metabolismo , Metilmalonil-CoA Descarboxilase/genética , Succinatos/metabolismo , Ácido Succínico/metabolismo
15.
Appl Environ Microbiol ; 87(6)2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33397698

RESUMO

Nicotine and nicotinic acid (NA) are both considered to be representatives of N-heterocyclic aromatic compounds, and their degradation pathways have been revealed in Pseudomonas species. However, the cooccurrence of these two pathways has only been observed in Pseudomonas sp. strain JY-Q. The nicotine pyrrolidine catabolism pathway of strain JY-Q consists of the functional modules Nic1, Spm, and Nic2. The module enzyme, 3-succinoylpyridine monooxygenase (Spm), catalyzes transformation of 3-succinoyl-pyridine (SP) to 6-hydroxy-3-succinoyl-pyridine (HSP). There exist two homologous but not identical Spm enzymes (namely, Spm1 and Spm2) in JY-Q. However, when spm1 and spm2 were both in-frame deleted, the mutant still grew well in basic salt medium (BSM) supplemented with nicotine as the sole carbon/nitrogen nutrition, suggesting that there exists an alternative pathway responsible for SP catabolism in JY-Q. NicAB, an enzyme accounting for NA hydroxylation, contains reorganized domains similar to those of Spm. When the JY-Q_nicAB gene (nicAB in strain JY-Q) was introduced into another Pseudomonas strain, one that is unable to degrade NA, the resultant recombinant strain exhibited the ability to transform SP to HSP, but without the ability to metabolize NA. Here, we conclude that NicAB in strain JY-Q exhibits an additional role in SP transformation. The other genes in the NA cluster, NicXDFE (Nic2 homolog), then also exhibit a role in subsequent HSP metabolism for energy yield. This finding also suggests that the cooccurrence of nicotine and NA degradation genes in strain JY-Q represents an advantage for JY-Q, making it more effective and flexible for the degradation of nicotine.IMPORTANCE 3-Succinoyl-pyridine (SP) and 6-hydroxy-3-succinoyl-pyridine (HSP) are both valuable chemical precursors to produce insecticides and hypotensive agents. SP and HSP could be renewable through the nicotine microbial degradation pathway, in which 3-succinoylpyridine monooxygenases (Spm) account for transforming SP into HSP in Pseudomonas sp. strain JY-Q. However, when two homologous Spm genes (spm1 and spm2) were knocked out, the mutant retained the ability to degrade nicotine. Thus, in addition to Spm, JY-Q should have an alternative pathway for SP conversion. In this research, we showed that JY-Q_NicAB was responsible for this alternative SP conversion. Both of the primary functions for nicotinic acid dehydrogenation and the additional function for SP metabolism were detected in a recombinant strain harboring JY-Q_NicAB. As a result, both nicotinic acid and nicotine degradation pathways in JY-Q contribute to its remarkable nicotine tolerance and nicotine degradation availability. These findings also provide one more metabolic engineering strategy for accumulation for value-added intermediates.


Assuntos
Proteínas de Bactérias/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Pseudomonas/enzimologia , Piridinas/metabolismo , Succinatos/metabolismo , Nicotina/metabolismo , Pseudomonas/genética
16.
J Appl Microbiol ; 130(6): 1972-1980, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33064909

RESUMO

AIM: Aspergillus niger S17-5 produces two alkylitaconic acids, 9-hydroxyhexylitaconic acid (9-HHIA) and 10-hydroxyhexylitaconic acid (10-HHIA), which have cytotoxic and polymer building block properties. In this study, we characterized the production of 9-HHIA and 10-HHIA by addition of their expected precursor, caprylic acid, to a culture of A. niger S17-5, and demonstrated batch fermentation of 9-HHIA and 10-HHIA in a jar fermenter with DO-stat. METHODS AND RESULTS: Production titres of 9-HHIA and 10-HHIA from 3% glucose in a flask after 25 days cultivation were 0·35 and 1·01 g l-1 respectively. Addition of 0·22 g l-1 of caprylic acid to a suspension of resting cells of A. niger S17-5 led to 32% enhancement of total 9-HHIA and 10-HHIA production compared to no addition. No enhancement of the production of 9-HHIA or 10-HHIA by the addition of oxaloacetic acid was observed. Addition of caprylic acid to the culture at mid-growth phase was more suitable for 9-HHIA and 10-HHIA production due to less cell growth inhibition by caprylic acid. DO-stat batch fermentation with 3% glucose and 14·4 g l-1 of caprylic acid in a 1·5 l jar fermenter resulted in the production titres of 9-HHIA and 10-HHIA being 0·48 and 1·54 g l-1 respectively after 10 days of cultivation. CONCLUSIONS: Addition of caprylic acid to the culture of A. niger S17-5 enhances 9-HHIA and 10-HHIA production. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that 9-HHIA and 10-HHIA are synthesized with octanoyl-CoA derived from caprylic acid, and that the supply of octanoyl-CoA is a rate-limiting step in 9-HHIA and 10-HHIA production. To the best of our knowledge, this is the first report regarding the fermentation of naturally occurring itaconic acid derivatives in a jar fermenter.


Assuntos
Aspergillus niger/metabolismo , Caprilatos/metabolismo , Succinatos/metabolismo , Aspergillus niger/efeitos dos fármacos , Aspergillus niger/crescimento & desenvolvimento , Reatores Biológicos , Caprilatos/análise , Caprilatos/farmacologia , Fermentação , Glucose/análise , Glucose/metabolismo , Succinatos/análise , Succinatos/química
17.
Int J Mol Sci ; 21(22)2020 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-33233586

RESUMO

Ecological concerns have recently led to the increasing trend to upgrade carbon contained in waste streams into valuable chemicals. One of these components is acetate. Its microbial upgrading is possible in various species, with Escherichia coli being the best-studied. Several chemicals derived from acetate have already been successfully produced in E. coli on a laboratory scale, including acetone, itaconic acid, mevalonate, and tyrosine. As acetate is a carbon source with a low energy content compared to glucose or glycerol, energy- and redox-balancing plays an important role in acetate-based growth and production. In addition to the energetic challenges, acetate has an inhibitory effect on microorganisms, reducing growth rates, and limiting product concentrations. Moreover, extensive metabolic engineering is necessary to obtain a broad range of acetate-based products. In this review, we illustrate some of the necessary energetic considerations to establish robust production processes by presenting calculations of maximum theoretical product and carbon yields. Moreover, different strategies to deal with energetic and metabolic challenges are presented. Finally, we summarize ways to alleviate acetate toxicity and give an overview of process engineering measures that enable sustainable acetate-based production of value-added chemicals.


Assuntos
Acetatos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Engenharia Metabólica/métodos , Poluentes Químicos da Água/metabolismo , Acetona/metabolismo , Biocatálise , Biocombustíveis , Carbono/metabolismo , Metabolismo Energético , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Humanos , Microbiologia Industrial/métodos , Redes e Vias Metabólicas/genética , Ácido Mevalônico/metabolismo , Succinatos/metabolismo , Tirosina/biossíntese
18.
Sci Immunol ; 5(52)2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097591

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease in which airway macrophages (AMs) play a key role. Itaconate has emerged as a mediator of macrophage function, but its role during fibrosis is unknown. Here, we reveal that itaconate is an endogenous antifibrotic factor in the lung. Itaconate levels are reduced in bronchoalveolar lavage, and itaconate-synthesizing cis-aconitate decarboxylase expression (ACOD1) is reduced in AMs from patients with IPF compared with controls. In the murine bleomycin model of pulmonary fibrosis, Acod1-/- mice develop persistent fibrosis, unlike wild-type (WT) littermates. Profibrotic gene expression is increased in Acod1-/- tissue-resident AMs compared with WT, and adoptive transfer of WT monocyte-recruited AMs rescued mice from disease phenotype. Culture of lung fibroblasts with itaconate decreased proliferation and wound healing capacity, and inhaled itaconate was protective in mice in vivo. Collectively, these data identify itaconate as critical for controlling the severity of lung fibrosis, and targeting this pathway may be a viable therapeutic strategy.


Assuntos
Carboxiliases/metabolismo , Fibrose Pulmonar Idiopática/imunologia , Macrófagos Alveolares/imunologia , Succinatos/metabolismo , Administração por Inalação , Transferência Adotiva/métodos , Adulto , Idoso , Animais , Bleomicina/administração & dosagem , Bleomicina/toxicidade , Líquido da Lavagem Broncoalveolar/imunologia , Broncoscopia , Estudos de Casos e Controles , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fibroblastos , Voluntários Saudáveis , Humanos , Hidroliases/genética , Hidroliases/metabolismo , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/diagnóstico , Fibrose Pulmonar Idiopática/terapia , Pulmão/citologia , Pulmão/imunologia , Pulmão/patologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/transplante , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Cultura Primária de Células , Índice de Gravidade de Doença , Succinatos/administração & dosagem , Succinatos/imunologia
19.
Cell ; 183(1): 62-75.e17, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32946811

RESUMO

In response to skeletal muscle contraction during exercise, paracrine factors coordinate tissue remodeling, which underlies this healthy adaptation. Here we describe a pH-sensing metabolite signal that initiates muscle remodeling upon exercise. In mice and humans, exercising skeletal muscle releases the mitochondrial metabolite succinate into the local interstitium and circulation. Selective secretion of succinate is facilitated by its transient protonation, which occurs upon muscle cell acidification. In the protonated monocarboxylic form, succinate is rendered a transport substrate for monocarboxylate transporter 1, which facilitates pH-gated release. Upon secretion, succinate signals via its cognate receptor SUCNR1 in non-myofibrillar cells in muscle tissue to control muscle-remodeling transcriptional programs. This succinate-SUCNR1 signaling is required for paracrine regulation of muscle innervation, muscle matrix remodeling, and muscle strength in response to exercise training. In sum, we define a bioenergetic sensor in muscle that utilizes intracellular pH and succinate to coordinate tissue adaptation to exercise.


Assuntos
Músculo Esquelético/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ácido Succínico/metabolismo , Animais , Humanos , Concentração de Íons de Hidrogênio , Inflamação/metabolismo , Camundongos , Mitocôndrias/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Contração Muscular , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais , Succinatos/metabolismo , Simportadores/metabolismo
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