RESUMO
Anaplerotic reactions catalyzed by pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC) have important roles in the production of l-lysine to replenish oxaloacetic acid (OAA) in Corynebacterium glutamicum. However, the relative contributions of these enzymes to l-lysine production in C. glutamicum are not fully understood. In this study, using a parent strain (P) carrying a feedback inhibition-resistant aspartokinase with the T311I mutation, we constructed a PC gene-deleted mutant strain (PΔPC) and a PEPC gene-deleted mutant strain (PΔPEPC). Although the growth of both mutant strains was comparable to the growth of strain P, the maximum l-lysine production in strains PΔPC and PΔPEPC decreased by 14% and 49%, respectively, indicating that PEPC strongly contributed to OAA supply. l-Lysine production in strain PΔPC slightly decreased during the logarithmic phase, while production during the early stationary phase was comparable to production in strain P. By contrast, strain PΔPEPC produced l-lysine in an amount comparable to the production of strain P during the logarithmic phase; l-lysine production after the early stationary phase was completely stopped in strain PΔPEPC. These results indicate that OAA is supplied by both PC and PEPC during the logarithmic phase, while only PEPC can continuously supply OAA after the logarithmic phase.
Assuntos
Corynebacterium glutamicum , Lisina , Fosfoenolpiruvato Carboxilase , Piruvato Carboxilase , Corynebacterium glutamicum/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/enzimologia , Lisina/metabolismo , Lisina/biossíntese , Piruvato Carboxilase/metabolismo , Piruvato Carboxilase/genética , Fosfoenolpiruvato Carboxilase/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Ácido Oxaloacético/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Mutação , Aspartato Quinase/metabolismo , Aspartato Quinase/genética , Deleção de GenesRESUMO
Tumor necrosis factor (TNF) has well-established roles in neuroinflammatory disorders, but the effect of TNF on the biochemistry of brain cells remains poorly understood. Here, we microinjected TNF into the brain to study its impact on glial and neuronal metabolism (glycolysis, pentose phosphate pathway, citric acid cycle, pyruvate dehydrogenase, and pyruvate carboxylase pathways) using 13C NMR spectroscopy on brain extracts following intravenous [1,2-13C]-glucose (to probe glia and neuron metabolism), [2-13C]-acetate (probing astrocyte-specific metabolites), or [3-13C]-lactate. An increase in [4,5-13C]-glutamine and [2,3-13C]-lactate coupled with a decrease in [4,5-13C]-glutamate was observed in the [1,2-13C]-glucose-infused animals treated with TNF. As glutamine is produced from glutamate by astrocyte-specific glutamine synthetase the increase in [4,5-13C]-glutamine reflects increased production of glutamine by astrocytes. This was confirmed by infusion with astrocyte substrate [2-13C]-acetate. As lactate is metabolized in the brain to produce glutamate, the simultaneous increase in [2,3-13C]-lactate and decrease in [4,5-13C]-glutamate suggests decreased lactate utilization, which was confirmed using [3-13C]-lactate as a metabolic precursor. These results suggest that TNF rearranges the metabolic network, disrupting the energy supply chain perturbing the glutamine-glutamate shuttle between astrocytes and the neurons. These insights pave the way for developing astrocyte-targeted therapeutic strategies aimed at modulating effects of TNF to restore metabolic homeostasis in neuroinflammatory disorders.
Assuntos
Astrócitos , Encéfalo , Ácido Glutâmico , Glutamina , Neurônios , Fator de Necrose Tumoral alfa , Animais , Astrócitos/metabolismo , Astrócitos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Ratos , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13/métodos , Ácido Láctico/metabolismo , Glucose/metabolismo , Masculino , Ciclo do Ácido Cítrico/efeitos dos fármacos , Isótopos de Carbono , Glicólise/efeitos dos fármacos , Acetatos/farmacologia , Acetatos/metabolismo , Piruvato Carboxilase/metabolismo , Via de Pentose Fosfato/efeitos dos fármacosRESUMO
BACKGROUND: Metabolic plasticity mediates breast cancer survival, growth, and immune evasion during metastasis. However, how tumor cell metabolism is influenced by and feeds back to regulate breast cancer progression are not fully understood. We identify hypoxia-mediated suppression of pyruvate carboxylase (PC), and subsequent induction of lactate production, as a metabolic regulator of immunosuppression. METHODS: We used qPCR, immunoblot, and reporter assays to characterize repression of PC in hypoxic primary tumors. Steady state metabolomics were used to identify changes in metabolite pools upon PC depletion. In vivo tumor growth and metastasis assays were used to evaluate the impact of PC manipulation and pharmacologic inhibition of lactate transporters. Immunohistochemistry, flow cytometry, and global gene expression analyzes of tumor tissue were employed to characterize the impact of PC depletion on tumor immunity. RESULTS: PC is essential for metastatic colonization of the lungs. In contrast, depletion of PC in tumor cells promotes primary tumor growth. This effect was only observed in immune competent animals, supporting the hypothesis that repression of PC can suppress anti-tumor immunity. Exploring key differences between the pulmonary and mammary environments, we demonstrate that hypoxia potently downregulated PC. In the absence of PC, tumor cells produce more lactate and undergo less oxidative phosphorylation. Inhibition of lactate metabolism was sufficient to restore T cell populations to PC-depleted mammary tumors. CONCLUSIONS: We present a dimorphic role for PC in primary mammary tumors vs. pulmonary metastases. These findings highlight a key contextual role for PC-directed lactate production as a metabolic nexus connecting hypoxia and antitumor immunity.
Assuntos
Neoplasias da Mama , Piruvato Carboxilase , Piruvato Carboxilase/metabolismo , Piruvato Carboxilase/genética , Animais , Feminino , Camundongos , Humanos , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Ácido Láctico/metabolismo , Regulação Neoplásica da Expressão Gênica , Hipóxia Celular , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Tolerância ImunológicaRESUMO
We propose a detailed computational beta cell model that emphasizes the role of anaplerotic metabolism under glucose and glucose-glutamine stimulation. This model goes beyond the traditional focus on mitochondrial oxidative phosphorylation and ATP-sensitive K+ channels, highlighting the predominant generation of ATP from phosphoenolpyruvate in the vicinity of KATP channels. It also underlines the modulatory role of H2O2 as a signaling molecule in the first phase of glucose-stimulated insulin secretion. In the second phase, the model emphasizes the critical role of anaplerotic pathways, activated by glucose stimulation via pyruvate carboxylase and by glutamine via glutamate dehydrogenase. It particularly focuses on the production of NADPH and glutamate as key enhancers of insulin secretion. The predictions of the model are consistent with empirical data, highlighting the complex interplay of metabolic pathways and emphasizing the primary role of glucose and the facilitating role of glutamine in insulin secretion. By delineating these crucial metabolic pathways, the model provides valuable insights into potential therapeutic targets for diabetes.
Assuntos
Glucose , Glutamina , Secreção de Insulina , Insulina , Modelos Biológicos , Glutamina/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Animais , Piruvato Carboxilase/metabolismo , Peróxido de Hidrogênio/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
This research aimed to explore the potential influence of mitochondria on the rate of anaerobic glycolysis. We hypothesized that mitochondria could reduce the rate of anaerobic glycolysis and pH decline by metabolizing a portion of glycolytic pyruvate. We utilized an in vitro model and incorporated CPI-613 and Avidin to inhibit pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC), respectively. Four treatments were tested: 400 µM CPI-613, 1.5 U/ml Avidin, 400 µM CPI-613 + 1.5 U/ml Avidin, or control. Glycolytic metabolites and pH of the in vitro model were evaluated throughout a 1440-min incubation period. CPI-613-containing treatments, with or without Avidin, decreased pH levels and increased glycogen degradation and lactate accumulation compared to the control and Avidin treatments (P < 0.05), indicating increased glycolytic flux. In a different experiment, two treatments, 400 µM CPI-613 or control, were employed to track the fates of pyruvate using [13C6]glucose. CPI-613 reduced the contribution of glucose carbon to tricarboxylic acid cycle intermediates compared to control (P < 0.05). To test whether the acceleration of acidification in reactions containing CPI-613 was due to an increase in the activity of key enzymes of glycogenolysis and glycolysis, we evaluated the activities of glycogen phosphorylase, phosphofructokinase, and pyruvate kinase in the presence or absence of 400 µM CPI-613. The CPI-613 treatment did not elicit an alteration in the activity of these three enzymes. These findings indicate that inhibiting PDH increases the rate of anaerobic glycolysis and pH decline, suggesting that mitochondria are potential regulators of postmortem metabolism.
Assuntos
Glicogênio , Glicólise , Complexo Piruvato Desidrogenase , Animais , Anaerobiose , Glucose/metabolismo , Glicogênio/metabolismo , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Mitocôndrias/metabolismo , Mudanças Depois da Morte , Piruvato Carboxilase/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Ácido Pirúvico/metabolismo , SuínosRESUMO
Anemoside B4 (AB4), a triterpenoidal saponin from Pulsatilla chinensis, shows significant anti-inflammatory activity, and may be used for treating inflammatory bowel disease (IBD). Nevertheless, its application is limited due to its high molecular weight and pronounced water solubility. To discover new effective agents for treating IBD, we synthesized 28 AB4 derivatives and evaluated their cytotoxic and anti-inflammatory activities in vitro. Among them, A3-6 exhibited significantly superior anti-inflammatory activity compared to AB4. It showed a significant improvement in the symptoms of DSS-induced colitis in mice, with a notably lower oral effective dose compared to AB4. Furthermore, we discovered that A3-6 bound with pyruvate carboxylase (PC), then inhibited PC activity, reprogramming macrophage function, and alleviated colitis. These findings indicate that A3-6 is a promising therapeutic candidate for colitis, and PC may be a potential new target for treating colitis.
Assuntos
Anti-Inflamatórios , Colite , Piruvato Carboxilase , Saponinas , Animais , Humanos , Camundongos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Anti-Inflamatórios/química , Anti-Inflamatórios/síntese química , Colite/tratamento farmacológico , Colite/induzido quimicamente , Sulfato de Dextrana , Descoberta de Drogas , Camundongos Endogâmicos C57BL , Piruvato Carboxilase/antagonistas & inibidores , Piruvato Carboxilase/metabolismo , Células RAW 264.7 , Saponinas/farmacologia , Saponinas/química , Saponinas/uso terapêutico , Saponinas/síntese química , Relação Estrutura-AtividadeRESUMO
Reprogrammed metabolism is a hallmark of cancer, but notoriously difficult to target due to metabolic plasticity, especially in response to single metabolic interventions. Combining mTOR inhibitor everolimus and mitochondrial complex 1 inhibitor metformin results in metabolic synergy in in vitro models of triple-negative breast cancer. Here, we investigated whether the effect of this drug combination on tumor size is reflected in changes in tumor metabolism using [U-13C]glucose labeling in an MDA-MB-231 triple negative breast cancer xenograft model. The in vitro effects of everolimus and metformin treatment on oxidative phosphorylation and glycolysis reflected changes in 13C-labeling of metabolites in MDA-MB-231 cells. Treatment of MDA-MB-231 xenografts in SCID/Beige mice with everolimus resulted in slower tumor growth and reduced tumor size and tumor viability by 35%. Metformin treatment moderately inhibited tumor growth but did not enhance everolimus-induced effects. High serum levels of everolimus were reached, whereas levels of metformin were relatively low. Everolimus decreased TCA cycle metabolite labeling and inhibited pyruvate carboxylase activity. Metformin only caused a mild reduction in glycolytic metabolite labeling and did not affect pyruvate carboxylase activity or TCA cycle metabolite labeling. In conclusion, treatment with everolimus, but not metformin, decreased tumor size and viability. Furthermore, the efficacy of everolimus was reflected in reduced 13C-labeling of TCA cycle intermediates and reduced pyruvate carboxylase activity. By using in-depth analysis of drug-induced changes in glucose metabolism in combination with measurement of drug levels in tumor and plasma, effects of metabolically targeted drugs can be explained, and novel targets can be identified.
Assuntos
Neoplasias da Mama , Metformina , Animais , Camundongos , Humanos , Feminino , Everolimo/farmacologia , Glucose/metabolismo , Piruvato Carboxilase , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células , Linhagem Celular Tumoral , Camundongos SCID , Metformina/farmacologiaRESUMO
Acetyl-CoA carboxylase (ACC) promotes prandial liver metabolism by producing malonyl-CoA, a substrate for de novo lipogenesis and an inhibitor of CPT-1-mediated fat oxidation. We report that inhibition of ACC also produces unexpected secondary effects on metabolism. Liver-specific double ACC1/2 knockout (LDKO) or pharmacologic inhibition of ACC increased anaplerosis, tricarboxylic acid (TCA) cycle intermediates, and gluconeogenesis by activating hepatic CPT-1 and pyruvate carboxylase flux in the fed state. Fasting should have marginalized the role of ACC, but LDKO mice maintained elevated TCA cycle intermediates and preserved glycemia during fasting. These effects were accompanied by a compensatory induction of proteolysis and increased amino acid supply for gluconeogenesis, which was offset by increased protein synthesis during feeding. Such adaptations may be related to Nrf2 activity, which was induced by ACC inhibition and correlated with fasting amino acids. The findings reveal unexpected roles for malonyl-CoA synthesis in liver and provide insight into the broader effects of pharmacologic ACC inhibition.
Assuntos
Acetil-CoA Carboxilase , Aminoácidos , Gluconeogênese , Fígado , Malonil Coenzima A , Camundongos Knockout , Oxirredução , Animais , Malonil Coenzima A/metabolismo , Fígado/metabolismo , Acetil-CoA Carboxilase/metabolismo , Camundongos , Aminoácidos/metabolismo , Masculino , Piruvato Carboxilase/metabolismo , Ciclo do Ácido Cítrico , Ácido Pirúvico/metabolismo , Camundongos Endogâmicos C57BL , Jejum/metabolismo , Carnitina O-Palmitoiltransferase/metabolismoRESUMO
BACKGROUND: Drug-resistant epilepsy is a common condition in patients with brain neoplasms. The pathogenesis of tumor-associated seizures is poorly understood. Among the possible pathogenetic mechanisms, the increase in glutamate concentration has been proposed. Glutamate transporters, glutamine synthetase and pyruvate carboxylase are involved in maintaining the physiological concentration of glutamate in the intersynaptic spaces. In our previous research on angiocentric gliomas, we demonstrated that all tumors lacked the expression of the main glutamate transporter EAAT2, while the expression of glutamine synthetase and pyruvate carboxylase was mostly preserved. METHODS: In the present study, we evaluated the immunohistochemical expression of EAAT2, glutamine synthetase and pyruvate carboxylase in a heterogeneous series of 25 long-term epilepsy-associated tumors (10 dysembryoplastic neuroepithelial tumors, 7 gangliogliomas, 3 subependymal giant cell astrocytomas, 3 rosette forming glioneuronal tumors, 1 diffuse astrocytoma MYB- or MYBL1-altered and 1 angiocentric glioma). In order to evaluate the incidence of variants in the SLC1A2 gene, encoding EAAT2, in a large number of central nervous system tumors we also queried the PedcBioPortal. RESULTS: EAAT2 protein expression was lost in 9 tumors (36 %: 3 dysembryoplastic neuroepithelial tumors, 1 ganglioglioma, 3 subependymal giant cell astrocytomas, 1 diffuse astrocytoma MYB- or MYBL1-altered and 1 angiocentric glioma). Glutamine synthetase protein expression was completely lost in 2 tumors (8 %; 1 ganglioglioma and 1 diffuse astrocytoma MYB- or MYBL1-altered). All tumors of our series but rosette forming glioneuronal tumors (in which neurocytic cells were negative) were diffusely positive for pyruvate carboxylase. Consultation of the PedcBioPortal revealed that of 2307 pediatric brain tumors of different histotype and grade, 20 (< 1%) had variants in the SLC1A2 gene. Among the SLC1A2-mutated tumors, there were no angiocentric gliomas or other LEATs CONCLUSIONS: In conclusion, unlike angiocentric gliomas where the EAAT2 loss is typical and constant, the current study shows the loss of EAAT2 expression only in a fraction of the LEATs. In these cases, we may hypothesize some possible epileptogenic role of the EAAT2 loss. The retained expression of pyruvate carboxylase may contribute to determining a pathological glutamate excess unopposed by glutamine synthetase that resulted expressed to a variable extent in the majority of the tumors. Furthermore, we can assume that the EAAT2 loss in brain tumors in general and in LEATs in particular is more conceivably epigenetic.
Assuntos
Astrocitoma , Neoplasias Encefálicas , Epilepsia , Ganglioglioma , Glioma , Neoplasias Neuroepiteliomatosas , Criança , Humanos , Astrocitoma/complicações , Astrocitoma/metabolismo , Astrocitoma/patologia , Neoplasias Encefálicas/metabolismo , Epilepsia/etiologia , Ganglioglioma/metabolismo , Glioma/genética , Glutamato-Amônia Ligase , Glutamatos , Piruvato Carboxilase , Convulsões/complicaçõesRESUMO
OBJECTIVES: Colitis is a global disease usually accompanied by intestinal epithelial damage and intestinal inflammation, and an increasing number of studies have found natural products to be highly effective in treating colitis. Anemoside B4 (AB4), an abundant saponin isolated from Pulsatilla chinensis (Bunge), which was found to have strong anti-inflammatory activity. However, the exact molecular mechanisms and direct targets of AB4 in the treatment of colitis remain to be discovered. METHODS: The anti-inflammatory activities of AB4 were verified in LPS-induced cell models and 2, 4, 6-trinitrobenzene sulfonic (TNBS) or dextran sulfate sodium (DSS)-induced colitis mice and rat models. The molecular target of AB4 was identified by affinity chromatography analysis using chemical probes derived from AB4. Experiments including proteomics, molecular docking, biotin pull-down, surface plasmon resonance (SPR), and cellular thermal shift assay (CETSA) were used to confirm the binding of AB4 to its molecular target. Overexpression of pyruvate carboxylase (PC) and PC agonist were used to study the effects of PC on the anti-inflammatory and metabolic regulation of AB4 in vitro and in vivo. RESULTS: AB4 not only significantly inhibited LPS-induced NF-κB activation and increased ROS levels in THP-1 cells, but also suppressed TNBS/DSS-induced colonic inflammation in mice and rats. The molecular target of AB4 was identified as PC, a key enzyme related to fatty acid, amino acid and tricarboxylic acid (TCA) cycle. We next demonstrated that AB4 specifically bound to the His879 site of PC and altered the protein's spatial conformation, thereby affecting the enzymatic activity of PC. LPS activated NF-κB pathway and increased PC activity, which caused metabolic reprogramming, while AB4 reversed this phenomenon by inhibiting the PC activity. In vivo studies showed that diisopropylamine dichloroacetate (DADA), a PC agonist, eliminated the therapeutic effects of AB4 by changing the metabolic rearrangement of intestinal tissues in colitis mice. CONCLUSION: We identified PC as a direct cellular target of AB4 in the modulation of inflammation, especially colitis. Moreover, PC/pyruvate metabolism/NF-κB is crucial for LPS-driven inflammation and oxidative stress. These findings shed more light on the possibilities of PC as a potential new target for treating colitis.
Assuntos
Colite , Saponinas , Ratos , Camundongos , Animais , Piruvato Carboxilase/metabolismo , NF-kappa B/metabolismo , Lipopolissacarídeos/farmacologia , Simulação de Acoplamento Molecular , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/metabolismo , Inflamação/metabolismo , Saponinas/farmacologia , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Macrófagos/metabolismo , Sulfato de Dextrana/efeitos adversos , Sulfato de Dextrana/metabolismo , Camundongos Endogâmicos C57BL , Modelos Animais de DoençasRESUMO
tRFs and tiRNAs (tRNA-derived fragments) are an emerging class of small noncoding RNAs produced by the precise shearing of tRNAs in response to specific stimuli. They have been reported to regulate the pathological processes of numerous human cancers. However, the biofunction of tRFs and tiRNAs in the development and progression of papillary thyroid cancer (PTC) has not been reported yet. In this study, we aimed to explore the biological roles of tRFs and tiRNAs in PTC and discovered that a novel 5'tRNA-derived fragment called tRF-1:30-Gly-CCC-3 (tRF-30) was markedly down-regulated in PTC tissues and cell lines. Functionally, tRF-30 inhibited the proliferation and invasion of PTC cells. Mechanistically, tRF-30 directly bound to the biotin-dependent enzyme pyruvate carboxylase (PC), downregulated its protein level, interfered with the TCA cycle intermediate anaplerosis, and thus affected metabolic reprogramming and PTC progression. These findings revealed a novel regulatory mechanism for tRFs and a potential therapeutic target for PTC.
Assuntos
Piruvato Carboxilase , Neoplasias da Glândula Tireoide , Humanos , Piruvato Carboxilase/metabolismo , Neoplasias da Glândula Tireoide/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Linhagem CelularRESUMO
Background: Inborne errors of metabolism are a common cause of neonatal death. This study evaluated the acute early-onset metabolic derangement and death in two unrelated neonates. Methods: Whole-exome sequencing (WES), Sanger sequencing, homology modeling, and in silico bioinformatics analysis were employed to assess the effects of variants on protein structure and function. Results: WES revealed a novel homozygous variant, p.G303Afs*40 and p.R156P, in the pyruvate carboxylase (PC) gene of each neonate, which both were confirmed by Sanger sequencing. Based on the American College of Medical Genetics and Genomics guidelines, the p.G303Afs*40 was likely pathogenic, and the p.R156P was a variant of uncertain significance (VUS). Nevertheless, a known variant at position 156, the p.R156Q, was also a VUS. Protein secondary structure prediction showed changes in p.R156P and p.R156Q variants compared to the wild-type protein. However, p.G303Afs*40 depicted significant changes at C-terminal. Furthermore, comparing the interaction of wild-type and variant proteins with the ATP ligand during simulations, revealed a decreased affinity to the ATP in all the variants. Moreover, analysis of Single nucleotide polymorphism impacts on PC protein using Polyphen-2, SNAP2, FATHMM, and SNPs&GO servers predicted both R156P and R156Q as damaging variants. Likewise, free energy calculations demonstrated the destabilizing effect of both variants on PC. Conclusion: This study confirmed the pathogenicity of both variants and suggested them as a cause of type B Pyruvate carboxylase deficiency. The results of this study would provide the family with prenatal diagnosis and expand the variant spectrum in the PC gene,which is beneficial for geneticists and endocrinologists.
Assuntos
Doença da Deficiência de Piruvato Carboxilase , Humanos , Recém-Nascido , Feminino , Gravidez , Doença da Deficiência de Piruvato Carboxilase/genética , Piruvato Carboxilase/genética , Piruvato Carboxilase/química , Piruvato Carboxilase/metabolismo , Estrutura Secundária de Proteína , Trifosfato de AdenosinaRESUMO
Allosteric regulation of the essential anaplerotic enzyme, pyruvate carboxylase (PC), is vital for metabolic homeostasis. PC catalyzes the bicarbonate- and ATP-dependent carboxylation of pyruvate to form oxaloacetate. Dysregulation of PC activity can impact glucose and redox metabolism, which contributes to the pathogenicity of many diseases. To maintain homeostasis, PC is allosterically activated by acetyl-CoA and allosterically inhibited by l-aspartate. In this study, we further characterize the molecular basis of allosteric regulation in Staphylococcus aureus PC (SaPC) using slowly/nonhydrolyzable dethia analogues of acetyl-CoA and site-directed mutagenesis of residues at the biotin carboxylase homodimer interface. The dethia analogues fully activate SaPC but demonstrate significantly reduced binding affinities relative to acetyl-CoA. Residues Arg21, Lys46, and Glu418 of SaPC are located at the biotin carboxylase dimer interface and play a critical role in both allosteric activation and inhibition. A structure of R21A SaPC in complex with acetyl-CoA reveals an intact molecule of acetyl-CoA bound at the allosteric site, offering new molecular insights into the acetyl-CoA binding site. This study demonstrates that the biotin carboxylase domain dimer interface is a critical allosteric site in PC, serving as a convergence point for allosteric activation by acetyl-CoA and inhibition by l-aspartate.
Assuntos
Piruvato Carboxilase , Staphylococcus aureus , Sítio Alostérico , Piruvato Carboxilase/genética , Staphylococcus aureus/genética , Acetilcoenzima A , Ácido Aspártico , PolímerosRESUMO
A series of novel erianin analogues were designed and synthesized based on the bioisosterism principle by altering the two aromatic rings of erianin, the substituents on the rings and the linker between them. The analogues were evaluated as pyruvate carboxylase (PC) inhibitors in hepatocellular carcinoma cells. It was found that compounds 35 and 36, where fluorine replaces a hydroxyl group, exhibited higher activity than erianin (IC50 value of 17.30 nM) in liver cancer cells with IC50 values of 15.15 nM and 10.05 nM, respectively. Additionally, at a concentration of 10 nM, compounds 35 and 36 inhibited PC with inhibitory rates of 39.10% and 40.15%, respectively, exhibiting nearly identical inhibitory activity to erianin (inhibitory rate of 40.07%). Additionally, a computer simulation docking study demonstrated the basis for better interactions between the receptors and ligands. The fluorine atom of 35 can not only form hydrogen bonds with Lys-1043 (NHâ¯F, 2.04 Å), but also form fluorine bonds with the carbonyl groups of Lys-1043 (3.67 Å) and Glu-1046 (3.70 Å), due to the different orientations of the halogens on the B ring warhead. Conversely, the chlorine atom of 34 can only form alkyl hydrophobic interactions with the alkane chain in Lys-1043. Fluorinated compounds 35 and 36 also show better chemical stability and higher log P (clog P = 3.89 for 35 and 36) values than that of erianin (clog P = 3.07), and may be used as candidate compounds for further drug development.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Piruvato Carboxilase , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Simulação por Computador , Flúor , Halogênios , Neoplasias Hepáticas/tratamento farmacológico , Piruvato Carboxilase/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
Renal metabolism is essential for kidney functions and energy homeostasis in the body. The TCA cycle is the hub of metabolism, but the metabolic activities of the cycle in the kidney have rarely been investigated. This study is to assess metabolic processes at the level of the TCA cycle in the kidney based on isotopomer distributions in multiple metabolites. Isolated rat kidneys were perfused with media containing common substrates including lactate and alanine for an hour. One group of kidneys received [U-13 C3 ]lactate instead of natural abundance lactate while the other group received [U-13 C3 ]alanine instead of natural abundance alanine. Perfused kidneys and effluent were prepared for analysis using NMR spectroscopy. 13 C-labeling patterns in glutamate, fumarate, aspartate and succinate from the kidney extracts showed that pyruvate carboxylase and oxidative metabolism through the TCA cycle were comparably very active, but pyruvate cycling and pyruvate dehydrogenase were relatively less active. Isotopomer analyses with fumarate and malate from effluent, however, indicated that pyruvate carboxylase was much more active than the TCA cycle and other metabolic processes. The reverse equilibrium of oxaloacetate with four-carbon intermediates of the cycle was nearly complete (92%), based on the ratio of [2,3,4-13 C3 ]/[1,2,3-13 C3 ] in aspartate or malate. 13 C enrichment in glucose with 13 C-lactate supply was higher than that with 13 C-alanine. Isotopomer analyses with multiple metabolites (i.e., glutamate, fumarate, aspartate, succinate and malate) allowed us to assess relative metabolic processes in the TCA cycle in the kidney supplied with [U-13 C3 ]lactate. Data from the analytes were generally consistent, indicating highly active pyruvate carboxylase and oxidative metabolism through the TCA cycle. Different 13 C-labeling patterns in analytes from the kidney extracts versus effluent suggested metabolic compartmentalization.
Assuntos
Ciclo do Ácido Cítrico , Malatos , Ratos , Animais , Malatos/metabolismo , Piruvato Carboxilase/metabolismo , Ácido Aspártico/metabolismo , Glucose/metabolismo , Ácido Glutâmico/metabolismo , Ácido Pirúvico/metabolismo , Ácido Láctico , Succinatos , Alanina/metabolismo , Isótopos de Carbono/metabolismoRESUMO
Background: Pyruvate carboxylase (PC) is a key enzyme for gluconeogenesis. PC deficiency (PCD) is an extremely rare autosomal recessive metabolic disease and is divided into three types. Type B PCD is clinically featured by lactic acidosis, hyperammonemia, hypercitrullinemia, hypotonia, abnormal movement, and seizures. Case presentation: Here, we report the first case of type B PCD in China, presenting with intractable lactic acidosis shortly after birth. A compound heterozygous mutation in the PC gene was identified by whole-exome sequencing, NM_001040716.2: c.1154_1155del and c.152G>A, which were inherited from her asymptomatic parents, respectively. Furthermore, prenatal neuroradiological presentations including widened posterior horns of lateral ventricles, huge subependymal cysts, and increased biparietal diameter and head circumference were concerned. Symptomatic treatment was taken and the infant died at 26 days. Conclusion: To our knowledge, this is the minimum gestational age (22w5d) that's when the prenatal onset of the neuroradiologic phenotype of PCD was observed. PCD has a poor prognosis and lacks an effective treatment, so this paper is shared to highlight the importance of PCD prenatal diagnosis in the absence of family history.
Assuntos
Acidose Láctica , Doença da Deficiência de Piruvato Carboxilase , Gravidez , Feminino , Humanos , Doença da Deficiência de Piruvato Carboxilase/diagnóstico , Doença da Deficiência de Piruvato Carboxilase/genética , Piruvato Carboxilase/genética , Convulsões , Hipotonia MuscularRESUMO
Pyruvate carboxylase (PC) is a mitochondrial, biotin-containing enzyme catalyzing the ATP-dependent synthesis of oxaloacetate from pyruvate and bicarbonate, with a critical anaplerotic role in sustaining the brain metabolism. Based on the studies performed on animal models, PC expression was assigned to be glia-specific. To study PC distribution among human neural cells, we probed the cultured human astrocytes and brain sections with antibodies against PC. Additionally, we tested the importance of PC for the viability of cultured human astrocytes by applying the PC inhibitor 3-chloropropane-1,2-diol (CPD). Our results establish the expression of PC in mitochondria of human astrocytes in culture and brain tissue and also into a subpopulation of the neurons in situ. CPD negatively affected the viability of astrocytes in culture, which could be partially reversed by supplementing media with malate, 2-oxoglutarate, citrate, or pyruvate. The provided data estimates PC expression in human astrocytes and neurons in human brain parenchyma. Furthermore, the enzymatic activity of PC is vital for sustaining the viability of cultured astrocytes.
Assuntos
Astrócitos , Piruvato Carboxilase , Animais , Humanos , Piruvato Carboxilase/metabolismo , Astrócitos/metabolismo , Ácido Pirúvico/metabolismo , Encéfalo/metabolismo , Neurônios/metabolismoRESUMO
AIM: The purpose of this study was to investigate the function and mechanism of a novel peptide derived from adipose-derived stem cell-conditioned medium (ADSC-CM). METHODS: Mass spectrometry was applied to identify expressed peptides in ADSC-CM obtained at different time points. The cell counting kit-8 assay and quantitative reverse transcription polymerase chain reactions were performed to screen the functional peptides contained within ADSC-CM. RNA-seq, western blot, a back skin excisional model of BALB/c mice, the peptide pull-down assay, rescue experiments, untargeted metabolomics, and mixOmics analysis were performed to thoroughly understand the functional mechanism of selected peptide. RESULTS: A total of 93, 827, 1108, and 631 peptides were identified in ADSC-CM at 0, 24, 48, and 72 h of conditioning, respectively. A peptide named ADSCP2 (DENREKVNDQAKL) derived from ADSC-CM inhibited collagen and ACTA2 mRNAs in hypertrophic scar fibroblasts. Moreover, ADSCP2 facilitated wound healing and attenuated collagen deposition in a mouse model. ADSCP2 bound with the pyruvate carboxylase (PC) protein and inhibited PC protein expression. Overexpressing PC rescued the reduction in collagen and ACTA2 mRNAs caused by ADSCP2. Untargeted metabolomics identified 258 and 447 differential metabolites in the negative and positive mode, respectively, in the ADSCP2-treated group. The mixOmics analysis, which integrated RNA-seq and untargeted metabolomics data, provided a more holistic view of the functions of ADSCP2. CONCLUSION: Overall, a novel peptide derived from ADSC-CM, named ADSCP2, attenuated hypertrophic scar fibrosis in vitro and in vivo, and the novel peptide ADSCP2 might be a promising drug candidate for clinical scar therapy.
Assuntos
Cicatriz Hipertrófica , Camundongos , Animais , Cicatriz Hipertrófica/metabolismo , Cicatriz Hipertrófica/patologia , Piruvato Carboxilase , Peptídeos , Fibrose , Células-Tronco/metabolismo , Tecido Adiposo/metabolismoRESUMO
AIMS: Pyruvate carboxylase (PC) plays a key role in cancer cell metabolic reprogramming. Whether metabolic reprogramming and PC are related in PDAC is unclear. Here, the effect of PC expression on PDAC tumorigenesis and metabolic reprogramming were evaluated. MATERIALS AND METHODS: PC protein expression in PDAC and precancerous tissues was measured through immunohistochemistry. The maximum standardized uptake (SUVmax) of 18F-fluoro-2-deoxy-2-d-glucose (18F-FDG) in PDAC patient PET/CT scans before surgical resection was retrospectively determined. Stable PC-knockdown and PC-overexpressing cells were established using lentiviruses, and PDAC progression was assessed in vivo and in vitro. Lactate content, 18F-FDG cell uptake rate, mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in cells. RNA sequencing revealed and qPCR verified differentially expressed genes (DEGs) after PC knockdown. The signaling pathways involved were determined by Western blotting. KEY FINDINGS: PC was significantly upregulated in PDAC tissues vs. precancerous tissues. A high SUVmax correlated with PC upregulation. PC knockdown significantly inhibited PDAC progression. Lactate content, SUVmax, and ECAR significantly decreased after PC knockdown. Peroxisome proliferator-activated receptor gamma coactivator-one alpha (PGC-1α) was upregulated after PC knockdown; and PGC1a expression promoted AMPK phosphorylation to activate mitochondrial metabolism. Metformin significantly inhibited mitochondrial respiration after PC knockdown, further activated AMPK and downstream carnitine palmitoyltransferase 1A (CPT1A)-regulated fatty acid oxidation (FAO), and inhibited PDAC cells progression. SIGNIFICANCE: PDAC cell uptake of FDG was positively correlated with PC expression. PC promotes PDAC glycolysis, and reducing PC expression can increase PGC1a expression, activate AMPK, and restore metformin sensitivity.
Assuntos
Metformina , Neoplasias Pancreáticas , Lesões Pré-Cancerosas , Humanos , Metformina/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Piruvato Carboxilase/metabolismo , Fatores de Transcrição/metabolismo , Fluordesoxiglucose F18 , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Estudos Retrospectivos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Neoplasias PancreáticasRESUMO
Pyruvate carboxylase (PC) deficiency is a rare autosomal recessive mitochondrial neurometabolic disorder of energy deficit resulting in high morbidity and mortality, with limited therapeutic options. The PC homotetramer has a critical role in gluconeogenesis, anaplerosis, neurotransmitter synthesis, and lipogenesis. The main biochemical and clinical findings in PC deficiency (PCD) include lactic acidosis, ketonuria, failure to thrive, and neurological dysfunction. Use of the anaplerotic agent triheptanoin on a limited number of individuals with PCD has had mixed results. We expand on the potential utility of triheptanoin in PCD by examining the clinical, biochemical, molecular, and health-related quality-of-life (HRQoL) findings in a cohort of 12 individuals with PCD (eight with Type A and two each with Types B and C) treated with triheptanoin ranging for 6 days to about 7 years. The main endpoints were changes in blood lactate and HRQoL scores, but collection of useful data was limited to about half of subjects. An overall trend of lactate reduction with time on triheptanoin was noted, but with significant variability among subjects and only one subject reaching close to statistical significance for this endpoint. Parent reported HRQoL assessments with treatment showed mixed results, with some subjects showing no change, some improvement, and some worsening of overall scores. Subjects with buried amino acids in the pyruvate carboxyltransferase domain of PC that undergo destabilizing replacements may be more likely to respond (with lactate reduction or HRQoL improvement) to triheptanoin compared to those with replacements that disrupt tetramerization or subunit-subunit interface contacts. The reason for this difference is unclear and requires further validation. We observed significant variability but an overall trend of lactate reduction with time on triheptanoin and mixed parent reported outcome changes by HRQoL assessments for subjects with PCD on long-term triheptanoin. The mixed results noted with triheptanoin therapy in this study could be due to endpoint data limitation, variability of disease severity between subjects, limitation of the parent reported HRQoL tool, or subject genotype variability. Alternative designed trials and more study subjects with PCD will be needed to validate important observations from this work.