RESUMO
Aconitate decarboxylase-1 (ACOD1) is expressed by activated macrophages and generates itaconate that exerts anti-microbial and immunoregulatory effects. ACOD1-itaconate is essential for macrophage-mediated control of the intracellular pathogen Coxiella (C.) burnetii, which causes Q fever. Two isomers of itaconate, mesaconate and citraconate, have overlapping yet distinct activity on macrophage metabolism and inflammatory gene expression. Here, we found that all three isomers inhibited the growth of C. burnetii in axenic culture in ACCM-2 medium. However, only itaconate reduced C. burnetii replication efficiently in Acod1-/- macrophages. In contrast, addition of citraconate strongly increased C. burnetii replication in Acod1+/- macrophages, whereas mesaconate weakly enhanced bacterial burden in Acod1-/- macrophages. Analysis of intracellular isomers showed that exogenous citraconate and mesaconate inhibited the generation of itaconate by infected Acod1+/- macrophages. Uptake of added isomers into Acod1-/- macrophages was increased after infection for itaconate and mesaconate, but not for citraconate. Mesaconate, but not citraconate, competed with itaconate for uptake into macrophages. Taken together, inhibition of itaconate generation by macrophages and interference with the uptake of extracellular itaconate could be identified as potential mechanisms behind the divergent effects of citraconate and mesaconate on C. burnetii replication in macrophages or in axenic culture.
Assuntos
Cultura Axênica , Carboxiliases , Coxiella burnetii , Macrófagos , Succinatos , Coxiella burnetii/efeitos dos fármacos , Coxiella burnetii/crescimento & desenvolvimento , Succinatos/farmacologia , Animais , Macrófagos/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Carboxiliases/metabolismo , Camundongos Knockout , Febre Q/imunologia , Febre Q/microbiologia , Camundongos Endogâmicos C57BL , HidroliasesRESUMO
The industrial amino acid production workhorse, Corynebacterium glutamicum naturally produces low levels of 2,3,5,6-tetramethylpyrazine (TMP), a valuable flavor, fragrance, and commodity chemical. Here, we demonstrate TMP production (â¼0.8 g L-1) in C. glutamicum type strain ATCC13032 via overexpression of acetolactate synthase and/or α-acetolactate decarboxylase from Lactococcus lactis in CGXII minimal medium supplemented with 40 g L-1 glucose. This engineered strain also demonstrated growth and TMP production when the minimal medium was supplemented with up to 40% (v v-1) hydrolysates derived from ionic liquid-pretreated sorghum biomass. A key objective was to take the fully engineered strain developed in this study and interrogate medium parameters that influence the production of TMP, a critical post-strain engineering optimization. Design of experiments in a high-throughput plate format identified glucose, urea, and their ratio as significant components affecting TMP production. These two components were further optimized using response surface methodology. In the optimized CGXII medium, the engineered strain could produce up to 3.56 g L-1 TMP (4-fold enhancement in titers and 2-fold enhancement in yield, mol mol-1) from 80 g L-1 glucose and 11.9 g L-1 urea in shake flask batch cultivation. ONE-SENTENCE SUMMARY: Corynebacterium glutamicum was metabolically engineered to produce 2,3,5,6-tetramethylpyrazine followed by a design of experiments approach to optimize medium components for high-titer production.
Assuntos
Corynebacterium glutamicum , Meios de Cultura , Glucose , Engenharia Metabólica , Pirazinas , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Pirazinas/metabolismo , Engenharia Metabólica/métodos , Meios de Cultura/química , Glucose/metabolismo , Acetolactato Sintase/genética , Acetolactato Sintase/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Lactococcus lactis/enzimologia , Carboxiliases/genética , Carboxiliases/metabolismo , Ureia/metabolismoRESUMO
BACKGROUND: Serine residues in the protein backbone of heavily glycosylated proteoglycans are bound to glycosaminoglycans through a tetrasaccharide linker. UXS1 encodes UDP-glucuronate decarboxylase 1, which catalyzes synthesis of UDP-xylose, the donor of the first building block in the linker. Defects in other enzymes involved in formation of the tetrasaccharide linker cause so-called linkeropathies, characterized by short stature, radio-ulnar synostosis, decreased bone density, congenital contractures, dislocations, and more. METHODS: Whole exome sequencing was performed in a father and son who presented with a mild skeletal dysplasia, as well as the father's unaffected parents. Wild-type and mutant UXS1 were recombinantly expressed in Escherichia coli and purified. Enzyme activity was evaluated by LC-MS/MS. In vivo effects were studied using HeparinRed assay and metabolomics. RESULTS: The son had short long bones, normal epiphysis, and subtle metaphyseal changes especially in his legs. The likely pathogenic heterozygous variant NM_001253875.1(UXS1):c.557T>A p.(Ile186Asn) detected in the son was de novo in the father. Purified Ile186Asn-UXS1, in contrast to the wild-type, was not able to convert UDP-glucuronic acid to UDP-xylose. Plasma glycosaminoglycan levels were decreased in both son and father. CONCLUSION: This is the first report linking UXS1 to short-limbed short stature in humans.
Assuntos
Nanismo , Humanos , Masculino , Nanismo/genética , Nanismo/metabolismo , Nanismo/patologia , Carboxiliases/genética , Carboxiliases/metabolismo , Alelos , Fenótipo , Mutação , Adulto , LinhagemRESUMO
2-Phenylethanol, known for its rose-like odor and antibacterial activity, is synthesized via exogenous phenylpyruvate by the sequential reaction of phenylpyruvate decarboxylase (PDC) and aldehyde reductase. We first targeted ARO10, a phenylpyruvate decarboxylase gene from Saccharomyces cerevisiae, and identified a suitable aldehyde reductase gene. Co-expression of ARO10 and yahK in E. coli transformants yielded 1.1 g/L of 2-phenylethanol in batch culture. We hypothesized that there might be a bottleneck in PDC activity. The computer-based enzyme evolution was utilized to enhance production. The introduction of an amino acid substitution in ARO10 (ARO10 I544W) stabilized the aromatic ring of the phenylpyruvate substrate, increasing 2-phenylethanol yield 4.1-fold compared to wild-type ARO10. Cultivation of ARO10 I544W-expressing E. coli produced 2.5 g/L of 2-phenylethanol with a yield from glucose of 0.16 g/g after 72 h. This approach represents a significant advancement, achieving the highest yield of 2-phenylethanol from glucose using microbes to date.
Assuntos
Carboxiliases , Escherichia coli , Engenharia Metabólica , Álcool Feniletílico , Saccharomyces cerevisiae , Escherichia coli/metabolismo , Escherichia coli/genética , Álcool Feniletílico/metabolismo , Engenharia Metabólica/métodos , Carboxiliases/metabolismo , Carboxiliases/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Glucose/metabolismoRESUMO
Cytokine storm syndrome (CSS) is a life-threatening systemic inflammatory syndrome involving innate immune hyperactivity triggered by various therapies, infections, and autoimmune conditions. However, the potential interplay between innate immune cells is not fully understood. Here, using poly I:C and lipopolysaccharide (LPS)-induced cytokine storm models, a protective role of neutrophils through the modulation of macrophage activation was identified in a CSS model. Intravital imaging revealed neutrophil-derived extracellular vesicles (NDEVs) in the liver and spleen, which were captured by macrophages. NDEVs suppressed proinflammatory cytokine production by macrophages when cocultured in vitro or infused into CSS models. Metabolic profiling of macrophages treated with NDEV revealed elevated levels of the anti-inflammatory metabolite, itaconate, which is produced from cis-aconitate in the Krebs cycle by cis-aconitate decarboxylase (Acod1, encoded by Irg1). Irg1 in macrophages, but not in neutrophils, was critical for the NDEV-mediated anti-inflammatory effects. Mechanistically, NDEVs delivered miR-27a-3p, which suppressed the expression of Suclg1, the gene encoding the enzyme that metabolizes itaconate, thereby resulting in the accumulation of itaconate in macrophages. These findings demonstrated that neutrophil-to-macrophage communication mediated by extracellular vesicles is critical for promoting the anti-inflammatory reprogramming of macrophages in CSS and may have potential implications for the treatment of this fatal condition.
Assuntos
Síndrome da Liberação de Citocina , Vesículas Extracelulares , Macrófagos , Neutrófilos , Succinatos , Animais , Vesículas Extracelulares/metabolismo , Succinatos/metabolismo , Macrófagos/metabolismo , Macrófagos/imunologia , Neutrófilos/metabolismo , Neutrófilos/imunologia , Camundongos , Síndrome da Liberação de Citocina/metabolismo , Carboxiliases/metabolismo , Camundongos Endogâmicos C57BL , Comunicação Celular , MicroRNAs/metabolismo , MicroRNAs/genética , Citocinas/metabolismo , Masculino , Modelos Animais de Doenças , HidroliasesRESUMO
Neuronal ferroptosis plays a key role in neurologic deficits post intracerebral hemorrhage (ICH). However, the endogenous regulation of rescuing ferroptotic neurons is largely unexplored. Here, we analyzed the integrated alteration of metabolomic landscape after ICH using LC-MS and MALDI-TOF/TOF MS, and demonstrated that aconitate decarboxylase 1 (Irg1) and its product itaconate, a derivative of the tricarboxylic acid cycle, were protectively upregulated. Deficiency of Irg1 or depletion of neuronal Irg1 in striatal neurons was shown to exaggerate neuronal loss and behavioral dysfunction in an ICH mouse model using transgenic mice. Administration of 4-Octyl itaconate (4-OI), a cell-permeable itaconate derivative, and neuronal Irg1 overexpression protected neurons in vivo. In addition, itaconate inhibited ferroptosis in cortical neurons derived from mouse and human induced pluripotent stem cells in vitro. Mechanistically, we demonstrated that itaconate alkylated glutathione peroxidase 4 (GPx4) on its cysteine 66 and the modification allosterically enhanced GPx4's enzymatic activity by using a bioorthogonal probe, itaconate-alkyne (ITalk), and a GPx4 activity assay using phosphatidylcholine hydroperoxide. Altogether, our research suggested that Irg1/itaconate-GPx4 axis may be a future therapeutic strategy for protecting neurons from ferroptosis post ICH.
Assuntos
Ferroptose , Neurônios , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Succinatos , Animais , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Ferroptose/efeitos dos fármacos , Camundongos , Succinatos/farmacologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Humanos , Carboxiliases/metabolismo , Carboxiliases/genética , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Masculino , Camundongos Transgênicos , Modelos Animais de Doenças , HidroliasesRESUMO
iso-Orotate decarboxylase (IDCase), which is involved in the thymidine salvage pathway, has attracted considerable interest owing to its chemical similarity to a hypothetical DNA decarboxylase in mammals. Although valuable insights into the active DNA demethylation of 5-methyl-cytosine can be obtained from the decarboxylation mechanism of 5-carboxyl-uracil (5caU) catalyzed by IDCase, this mechanism remains under debate. In this study, the catalytic mechanism of 5caU decarboxylation by IDCase was studied using hybrid quantum mechanics/molecular mechanics (QM/MM) methodologies and density functional theory (DFT) calculations with a truncated model. The calculations supported a mechanism involving three sequential stages: activation of the 5caU substrate via proton transfer from an arginine (R262') to the carboxyl group of 5caU, formation of a tetrahedral intermediate, and decarboxylation of the tetrahedral intermediate to generate uracil as the product. The reaction pathways and structures obtained using the QM/MM and DFT methods coincided with each other. These simulations provided detailed insights into the unique mechanism of IDCase, clarifying various unresolved issues, such as the critical role of R262'. In addition, aspartate D323 was found to act as a general base in the tetrahedral intermediate formation step and a general acid in the later C-C bond cleavage step.
Assuntos
Teoria da Densidade Funcional , Descarboxilação , Simulação de Dinâmica Molecular , Teoria Quântica , Carboxiliases/química , Carboxiliases/metabolismo , Biocatálise , Orotidina-5'-Fosfato Descarboxilase/química , Orotidina-5'-Fosfato Descarboxilase/metabolismo , Uracila/química , Uracila/metabolismoRESUMO
Polycystic ovary syndrome (PCOS), an endocrine disorder afflicting 6-20% of women of reproductive age globally, has been linked to alterations in the gut microbiome. We previously showed that in PCOS, elevation of Bacteroides vulgatus in the gut microbiome was associated with altered bile acid metabolism. Here we show that B. vulgatus also induces a PCOS-like phenotype in female mice via an alternate mechanism independent of bile acids. We find that B. vulgatus contributes to PCOS-like symptoms through its metabolite agmatine, which is derived from arginine by arginine decarboxylase. Mechanistically, agmatine activates the farnesoid X receptor (FXR) pathway to subsequently inhibit glucagon-like peptide-1 (GLP-1) secretion by L cells, which leads to insulin resistance and ovarian dysfunction. Critically, the GLP-1 receptor agonist liraglutide and the arginine decarboxylase inhibitor difluoromethylarginine ameliorate ovarian dysfunction in a PCOS-like mouse model. These findings reveal that agmatine-FXR-GLP-1 signalling contributes to ovarian dysfunction, presenting a potential therapeutic target for PCOS management.
Assuntos
Agmatina , Microbioma Gastrointestinal , Síndrome do Ovário Policístico , Receptores Citoplasmáticos e Nucleares , Síndrome do Ovário Policístico/tratamento farmacológico , Síndrome do Ovário Policístico/metabolismo , Animais , Feminino , Camundongos , Agmatina/farmacologia , Agmatina/metabolismo , Agmatina/uso terapêutico , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismo , Microbioma Gastrointestinal/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Transdução de Sinais/efeitos dos fármacos , Modelos Animais de Doenças , Resistência à Insulina , Bacteroides/efeitos dos fármacos , Humanos , Carboxiliases/metabolismoRESUMO
Sepsis, a systemic inflammatory response triggered by infection, has a considerably high mortality rate. However, effective prevention and intervention measures against sepsis remain insufficient. Therefore, this study aimed to investigate the mechanisms underlying the protective properties of immune response gene-1 (IRG1) and 4-Octyl itaconate (OI) during acute liver damage in mice with sepsis. A sepsis mouse model was established to compare wild-type and IRG1-/- groups. The impact of IRG1/Itaconate on pro- and anti-inflammatory cytokines was evaluated using J774A.1 cells. IRG1/Itaconate substantially reduced pro-inflammatory cytokines and increased the release of anti-inflammatory cytokines. It reduced pathological damage to liver tissues, preserved normal liver function, decreased the release of reactive oxygen species (ROS) and LDH, and enhanced the GSH/GSSG ratio. Moreover, IRG1 and itaconic acid activated the Nrf2 signaling pathway, regulating the expression of its downstream antioxidative stress-related proteins. Additionally, they inhibited the activity of NLRP3 inflammatory vesicles to suppress the expression of macrophage-associated pyroptosis signaling molecules. Our findings demonstrate that IRG1/OI inhibits NLRP3 inflammatory vesicle activation and macrophage pyroptosis by modulating the Nrf2 signaling pathway, thereby attenuating acute liver injury in mice with sepsis. These findings could facilitate the clinical application of IRG1/Itaconate to prevent sepsis-induced acute liver injury.
Assuntos
Macrófagos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2 , Proteína 3 que Contém Domínio de Pirina da Família NLR , Piroptose , Sepse , Transdução de Sinais , Succinatos , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Succinatos/uso terapêutico , Succinatos/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Sepse/tratamento farmacológico , Sepse/complicações , Sepse/imunologia , Piroptose/efeitos dos fármacos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos Knockout , Fígado/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/imunologia , Linhagem Celular , Modelos Animais de Doenças , Citocinas/metabolismo , Hidroliases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Humanos , Carboxiliases/metabolismo , Carboxiliases/genéticaRESUMO
Cadaverine is a critical C5 monomer for the production of polyamides. Pyridoxal 5'-phosphate (PLP), as a crucial cofactor for the key enzyme lysine decarboxylase in the cadaverine biosynthesis pathway, has seen a persistent shortage, leading to limitations in cadaverine production. To address this issue, a dual-pathway strategy was implemented, synergistically enhancing both endogenous and heterologous PLP synthesis modules and resulting in improved PLP synthesis. Subsequently, a growth-stage-dependent molecular switch was introduced to balance the precursor competition between PLP synthesis and cell growth. Additionally, a PLP sensor-based negative feedback circuit was constructed by integrating a newly identified PLP-responsive promoter PygjH and an arabinose-regulated system, dynamically regulating the expression of the PLP synthetic genes and preventing excessive intracellular PLP accumulation. The optimal strain, L18, cultivated in the minimal medium AM1, demonstrated cadaverine production with a titer, yield, and productivity of 64.03 g/L, 0.23 g/g glucose, and 1.33 g/L/h, respectively. This represents the highest titer reported to date in engineered Escherichia coli by fed-batch fermentation in a minimal medium.
Assuntos
Cadaverina , Meios de Cultura , Escherichia coli , Engenharia Metabólica , Fosfato de Piridoxal , Cadaverina/metabolismo , Cadaverina/biossíntese , Fosfato de Piridoxal/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Meios de Cultura/química , Regiões Promotoras Genéticas , Carboxiliases/genética , Carboxiliases/metabolismoRESUMO
BACKGROUND: 1,5-pentanediol (1,5-PDO) is a linear diol with an odd number of methylene groups, which is an important raw material for polyurethane production. In recent years, the chemical methods have been predominantly employed for synthesizing 1,5-PDO. However, with the increasing emphasis on environmentally friendly production, it has been a growing interest in the biosynthesis of 1,5-PDO. Due to the limited availability of only three reported feasible biosynthesis pathways, we developed a new biosynthetic pathway to form a cell factory in Escherichia coli to produce 1,5-PDO. RESULTS: In this study, we reported an artificial pathway for the synthesis of 1,5-PDO from lysine with an integrated cofactor and co-substrate recycling and also evaluated its feasibility in E.coli. To get through the pathway, we first screened aminotransferases originated from different organisms to identify the enzyme that could successfully transfer two amines from cadaverine, and thus GabT from E. coli was characterized. It was then cascaded with lysine decarboxylase and alcohol dehydrogenase from E. coli to achieve the whole-cell production of 1,5-PDO from lysine. To improve the whole-cell activity for 1,5-PDO production, we employed a protein scaffold of EutM for GabT assembly and glutamate dehydrogenase was also validated for the recycling of NADPH and α-ketoglutaric acid (α-KG). After optimizing the cultivation and bioconversion conditions, the titer of 1,5-PDO reached 4.03 mM. CONCLUSION: We established a novel pathway for 1,5-PDO production through two consecutive transamination reaction from cadaverine, and also integrated cofactor and co-substrate recycling system, which provided an alternative option for the biosynthesis of 1,5-PDO.
Assuntos
Vias Biossintéticas , Escherichia coli , Escherichia coli/metabolismo , Escherichia coli/genética , Engenharia Metabólica/métodos , Glicóis/metabolismo , Lisina/metabolismo , Lisina/biossíntese , Álcool Desidrogenase/metabolismo , Transaminases/metabolismo , Transaminases/genética , Carboxiliases/metabolismoRESUMO
BACKGROUND: Obesity is the main risk factor leading to the development of various respiratory diseases, such as asthma and pulmonary hypertension. Pulmonary microvascular endothelial cells (PMVECs) play a significant role in the development of lung diseases. Aconitate decarboxylase 1 (Acod1) mediates the production of itaconate, and Acod1/itaconate axis has been reported to play a protective role in multiple diseases. However, the roles of Acod1/itaconate axis in the PMVECs of obese mice are still unclear. METHODS: mRNA-seq was performed to identify the differentially expressed genes (DEGs) between high-fat diet (HFD)-induced PMVECs and chow-fed PMVECs in mice (|log2 fold change| ≥ 1, p ≤ 0.05). Free fatty acid (FFA) was used to induce cell injury, inflammation and mitochondrial oxidative stress in mouse PMVECs after transfection with the Acod1 overexpressed plasmid or 4-Octyl Itaconate (4-OI) administration. In addition, we investigated whether the nuclear factor erythroid 2-like 2 (Nrf2) pathway was involved in the effects of Acod1/itaconate in FFA-induced PMVECs. RESULTS: Down-regulated Acod1 was identified in HFD mouse PMVECs by mRNA-seq. Acod1 expression was also reduced in FFA-treated PMVECs. Acod1 overexpression inhibited cell injury, inflammation and mitochondrial oxidative stress induced by FFA in mouse PMVECs. 4-OI administration showed the consistent results in FFA-treated mouse PMVECs. Moreover, silencing Nrf2 reversed the effects of Acod1 overexpression and 4-OI administration in FFA-treated PMVECs, indicating that Nrf2 activation was required for the protective effects of Acod1/itaconate. CONCLUSION: Our results demonstrated that Acod1/Itaconate axis might protect mouse PMVECs from FFA-induced injury, inflammation and mitochondrial oxidative stress via activating Nrf2 pathway. It was meaningful for the treatment of obesity-caused pulmonary microvascular endotheliopathy.
Assuntos
Carboxiliases , Células Endoteliais , Pulmão , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2 , Obesidade , Succinatos , Animais , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Camundongos , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Carboxiliases/metabolismo , Carboxiliases/genética , Obesidade/metabolismo , Obesidade/complicações , Masculino , Succinatos/farmacologia , Pulmão/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/irrigação sanguínea , Células Cultivadas , Microvasos/metabolismo , Microvasos/efeitos dos fármacos , Microvasos/patologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Dieta Hiperlipídica/efeitos adversos , Endotélio Vascular/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , HidroliasesRESUMO
Taurine (Tau) is a semiessential amino acid in mammals with preventive and therapeutic effects on several intestinal disorders. However, the exact function of taurine in ulcerative colitis (UC) is still largely unclear. In this study, we used two taurine-deficient mouse models (CSAD-/- and TauT-/- mice) to explore the influence of taurine on the progression of UC in both dextran sulfate sodium (DSS)-induced colitis and LPS-stimulated Caco-2 cells. We found that cysteine sulfinic acid decarboxylase (CSAD) and taurine transporter (TauT) expressions and taurine levels were markedly reduced in colonic tissues of mice treated with DSS. The CSAD and TauT knockouts exacerbated DSS-induced clinical symptoms and pathological damage and aggravated the intestinal barrier dysfunction and the colonic mucosal inflammatory response. Conversely, taurine pretreatment enhanced the intestinal barrier functions by increasing goblet cells and upregulating tight junction protein expression. Importantly, taurine bound with TLR4 and inhibited the TLR4/NF-κB pathway, ultimately reducing proinflammatory factors (TNF-α and IL-6) and oxidative stress. Our findings highlight the essential role of taurine in maintaining the intestinal barrier integrity and inhibiting intestinal inflammation, indicating that taurine is a promising supplement for colitis treatment.
Assuntos
Colite , Mucosa Intestinal , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B , Transdução de Sinais , Taurina , Receptor 4 Toll-Like , Animais , Taurina/farmacologia , Taurina/administração & dosagem , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Camundongos , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Colite/tratamento farmacológico , Colite/metabolismo , Colite/induzido quimicamente , Colite/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Células CACO-2 , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Sulfato de Dextrana/efeitos adversos , Carboxiliases/genética , Carboxiliases/metabolismo , Função da Barreira IntestinalRESUMO
Acid-induced arginine decarboxylase AdiA is a typical homo-oligomeric protein biosynthesizing alkaline nylon monomer putrescine. However, upon loss of the AdiA decamer oligomeric state at neutral and alkaline conditions the activity also diminishes, obstructing the whole-cell biosynthesis of alkaline putrescine. Here, a structure cohesion strategy is proposed to change the pH adaptation of AdiA to alkaline environments based on the rational engineering of meridional and latitudinal oligomerization interfaces. After integrating substitutions of E467K at the latitudinal interface and H736E at the meridional channel interface, the structural stability of AdiA decamer and its substrate transport efficiency at neutral and alkaline conditions are improved. Finally, E467K_H736E is well adapted to neutral and alkaline environments (pH 7.0-9.0), and its enzymatic activity is 35-fold higher than that of wild AdiA at pH 8.0. Using E467K_H736E in the putrescine synthesis pathway, the titer of putrescine is up to 128.9 g·L-1 with a conversion of 0.94 mol·mol-1 in whole-cell catalysis. Additionally, the neutral pH adaptation of lysine decarboxylase, with a decamer structure similar to AdiA, is also improved using this cohesion strategy, providing an option for pH-adaptation engineering of other oligomeric decarboxylases.
Assuntos
Carboxiliases , Escherichia coli , Putrescina , Carboxiliases/metabolismo , Carboxiliases/genética , Carboxiliases/química , Concentração de Íons de Hidrogênio , Escherichia coli/metabolismo , Escherichia coli/genética , Putrescina/metabolismoRESUMO
Carbohydrates and lipids provide the majority of substrates to fuel mitochondrial oxidative phosphorylation. Metabolic inflexibility, defined as an impaired ability to switch between these fuels, is implicated in a number of metabolic diseases. Here, we explore the mechanism by which physical inactivity promotes metabolic inflexibility in skeletal muscle. We developed a mouse model of sedentariness, small mouse cage (SMC), that, unlike other classic models of disuse in mice, faithfully recapitulated metabolic responses that occur in humans. Bioenergetic phenotyping of skeletal muscle mitochondria displayed metabolic inflexibility induced by physical inactivity, demonstrated by a reduction in pyruvate-stimulated respiration (JO2) in the absence of a change in palmitate-stimulated JO2. Pyruvate resistance in these mitochondria was likely driven by a decrease in phosphatidylethanolamine (PE) abundance in the mitochondrial membrane. Reduction in mitochondrial PE by heterozygous deletion of phosphatidylserine decarboxylase (PSD) was sufficient to induce metabolic inflexibility measured at the whole-body level, as well as at the level of skeletal muscle mitochondria. Low mitochondrial PE in C2C12 myotubes was sufficient to increase glucose flux toward lactate. We further implicate that resistance to pyruvate metabolism is due to attenuated mitochondrial entry via mitochondrial pyruvate carrier (MPC). These findings suggest a mechanism by which mitochondrial PE directly regulates MPC activity to modulate metabolic flexibility in mice.
Assuntos
Mitocôndrias Musculares , Músculo Esquelético , Fosfatidiletanolaminas , Ácido Pirúvico , Animais , Camundongos , Músculo Esquelético/metabolismo , Ácido Pirúvico/metabolismo , Mitocôndrias Musculares/metabolismo , Fosfatidiletanolaminas/metabolismo , Comportamento Sedentário , Masculino , Carboxiliases/metabolismo , Carboxiliases/genética , Camundongos Knockout , Estearoil-CoA DessaturaseRESUMO
Polyamines are polycations derived from amino acids that play an important role in proliferation and growth in almost all living cells. In Streptococcus pneumoniae (the pneumococcus), modulation of polyamine metabolism not only plays an important regulatory role in central metabolism, but also impacts virulence factors such as the capsule and stress responses that affect survival in the host. However, functional annotation of enzymes from the polyamine biosynthesis pathways in the pneumococcus is based predominantly on computational prediction. In this study, we cloned SP_0166, predicted to be a pyridoxal-dependent decarboxylase, from the Orn/Lys/Arg family pathway in S. pneumoniae TIGR4 and expressed and purified the recombinant protein. We performed biochemical characterization of the recombinant SP_0166 and confirmed the substrate specificity. For polyamine analysis, we developed a simultaneous quantitative method using hydrophilic interaction liquid chromatography (HILIC)-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. SP_0166 has apparent Km, kcat, and kcat/Km values of 11.3 mM, 715,053 min-1, and 63,218 min-1 mM-1, respectively, with arginine as a substrate at pH 7.5. We carried out inhibition studies of SP_0166 enzymatic activity with arginine as a substrate using chemical inhibitors DFMO and DFMA. DFMO is an irreversible inhibitor of ornithine decarboxylase activity, while DFMA inhibits arginine decarboxylase activity. Our findings confirm that SP_0166 is inhibited by DFMA and DFMO, impacting agmatine production. The use of arginine as a substrate revealed that the synthesis of putrescine by agmatinase and N-carbamoylputrescine by agmatine deiminase were both affected and inhibited by DFMA. This study provides experimental validation that SP_0166 is an arginine decarboxylase in pneumococci.
Assuntos
Carboxiliases , Streptococcus pneumoniae , Espectrometria de Massas em Tandem , Carboxiliases/metabolismo , Carboxiliases/genética , Carboxiliases/química , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/genética , Cromatografia Líquida de Alta Pressão , Especificidade por Substrato , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Poliaminas/metabolismo , CinéticaRESUMO
Glucocorticoids represent the mainstay of therapy for a broad spectrum of immune-mediated inflammatory diseases. However, the molecular mechanisms underlying their anti-inflammatory mode of action have remained incompletely understood1. Here we show that the anti-inflammatory properties of glucocorticoids involve reprogramming of the mitochondrial metabolism of macrophages, resulting in increased and sustained production of the anti-inflammatory metabolite itaconate and consequent inhibition of the inflammatory response. The glucocorticoid receptor interacts with parts of the pyruvate dehydrogenase complex whereby glucocorticoids provoke an increase in activity and enable an accelerated and paradoxical flux of the tricarboxylic acid (TCA) cycle in otherwise pro-inflammatory macrophages. This glucocorticoid-mediated rewiring of mitochondrial metabolism potentiates TCA-cycle-dependent production of itaconate throughout the inflammatory response, thereby interfering with the production of pro-inflammatory cytokines. By contrast, artificial blocking of the TCA cycle or genetic deficiency in aconitate decarboxylase 1, the rate-limiting enzyme of itaconate synthesis, interferes with the anti-inflammatory effects of glucocorticoids and, accordingly, abrogates their beneficial effects during a diverse range of preclinical models of immune-mediated inflammatory diseases. Our findings provide important insights into the anti-inflammatory properties of glucocorticoids and have substantial implications for the design of new classes of anti-inflammatory drugs.
Assuntos
Anti-Inflamatórios , Glucocorticoides , Inflamação , Macrófagos , Mitocôndrias , Succinatos , Animais , Feminino , Humanos , Masculino , Camundongos , Anti-Inflamatórios/farmacologia , Carboxiliases/metabolismo , Carboxiliases/antagonistas & inibidores , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ciclo do Ácido Cítrico/genética , Citocinas/imunologia , Citocinas/metabolismo , Glucocorticoides/farmacologia , Glucocorticoides/metabolismo , Hidroliases/deficiência , Hidroliases/genética , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Complexo Piruvato Desidrogenase/metabolismo , Receptores de Glucocorticoides/metabolismo , Succinatos/metabolismo , Ativação Enzimática/efeitos dos fármacosRESUMO
BACKGROUND: Spinal cord injury (SCI)-induced neuroinflammation and oxidative stress (OS) are crucial events causing neurological dysfunction. Aconitate decarboxylase 1 (ACOD1) and its metabolite itaconate (Ita) inhibit inflammation and OS by promoting alkylation of Keap1 to induce Nrf2 expression; however, it is unclear whether there is another pathway regulating their effects in inflammation-activated microglia after SCI. METHODS: Adult male C57BL/6 ACOD1-/- mice and their wild-type (WT) littermates were subjected to a moderate thoracic spinal cord contusion. The degree of neuroinflammation and OS in the injured spinal cord were assessed using qPCR, western blot, flow cytometry, immunofluorescence, and trans-well assay. We then employed immunoprecipitation-western blot, chromatin immunoprecipitation (ChIP)-PCR, dual-luciferase assay, and immunofluorescence-confocal imaging to examine the molecular mechanisms of ACOD1. Finally, the locomotor function was evaluated with the Basso Mouse Scale and footprint assay. RESULTS: Both in vitro and in vivo, microglia with transcriptional blockage of ACOD1 exhibited more severe levels of neuroinflammation and OS, in which the expression of p62/Keap1/Nrf2 was down-regulated. Furthermore, silencing ACOD1 exacerbated neurological dysfunction in SCI mice. Administration of exogenous Ita or 4-octyl itaconate reduced p62 phosphorylation. Besides, ACOD1 was capable of interacting with phosphorylated p62 to enhance Nrf2 activation, which in turn further promoted transcription of ACOD1. CONCLUSIONS: Here, we identified an unreported ACOD1-p62-Nrf2-ACOD1 feedback loop exerting anti-inflammatory and anti-OS in inflammatory microglia, and demonstrated the neuroprotective role of ACOD1 after SCI, which was different from that of endogenous and exogenous Ita. The present study extends the functions of ACOD1 and uncovers marked property differences between endogenous and exogenous Ita. KEY POINTS: ACOD1 attenuated neuroinflammation and oxidative stress after spinal cord injury. ACOD1, not itaconate, interacted with p-p62 to facilitate Nrf2 expression and nuclear translocation. Nrf2 was capable of promoting ACOD1 transcription in microglia.
Assuntos
Carboxiliases , Hidroliases , Microglia , Fator 2 Relacionado a NF-E2 , Traumatismos da Medula Espinal , Succinatos , Animais , Masculino , Camundongos , Carboxiliases/metabolismo , Carboxiliases/genética , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microglia/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Proteína Sequestossoma-1/metabolismo , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/complicações , Succinatos/farmacologia , Succinatos/metabolismoRESUMO
Aspergillus oryzae spores, when sprinkled onto steamed rice and allowed to propagate, are referred to as rice "koji." Agmatine, a natural polyamine derived from arginine through the action of arginine decarboxylase (ADC), is abundantly produced by solid state-cultivated rice koji of A. oryzae RIB40 under low pH conditions, despite the apparent absence of ADC orthologs in its genome. Mass spectrometry imaging revealed that agmatine was accumulated inside rice koji at low pH conditions, where arginine was distributed. ADC activity was predominantly observed in substrate mycelia and minimally in aerial mycelia. Natural ADC was isolated from solid state-cultivated A. oryzae rice koji containing substrate mycelia, using ammonium sulfate fractionation, ion exchange, and gel-filtration chromatography. The purified protein was subjected to sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE), and the detected peptide band was digested for identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The gene AO090102000327 of strain RIB40 was identified, previously annotated as phosphatidylserine decarboxylase (PSD), and encoded a 483-amino acid peptide. Recombinant protein encoded by AO090102000327 was expressed in Escherichia coli cells cultivated at 20°C, resulting in the detection of 49 kDa and 5 kDa peptides. The protein exhibited pyruvoyl-dependent decarboxylase activity, favoring arginine over ornithine and showing no activity with phosphatidylserine. The gene was designated Ao-adc1. Ao-ADC1 expression in rice koji at pH 4-6 was confirmed through western blotting using the anti-Ao-ADC1 serum. These findings indicate that Ao-adc1 encodes arginine decarboxylase involved in agmatine production.IMPORTANCEGene AO090102000327 in A. oryzae RIB40, previously annotated as a PSD, falls into a distinct clade when examining the phylogenetic distribution of PSDs. Contrary to the initial PSD annotation, our analysis indicates that the protein encoded by AO090102000327 is expressed in the substrate mycelia area of solid state-cultivated A. oryzae rice koji and functions as an arginine decarboxylase (ADC). The clade to which Ao-ADC1 belongs includes three other Ao-ADC1 paralogs (AO090103000445, AO090701000800, and AO090701000802) that presumably encode ADC rather than PSDs. Regarding PSD, AO090012000733 and AO090005001124 were speculated to be nonmitochondrial and mitochondrial PSDs in A. oryzae RIB40, respectively.