RESUMO
The phosphoenol pyruvate-oxaloacetate-pyruvate-derived amino acids (POP-AAs) comprise native intermediates in cellular metabolism, within which the phosphoenol pyruvate-oxaloacetate-pyruvate (POP) node is the switch point among the major metabolic pathways existing in most living organisms. POP-AAs have widespread applications in the nutrition, food, and pharmaceutical industries. These amino acids have been predominantly produced in Escherichia coli and Corynebacterium glutamicum through microbial fermentation. With the rapid increase in market requirements, along with the global food shortage situation, the industrial production capacity of these two bacteria has encountered two bottlenecks: low product conversion efficiency and high cost of raw materials. Aiming to push forward the update and upgrade of engineered strains with higher yield and productivity, this paper presents a comprehensive summarization of the fundamental strategy of metabolic engineering techniques around phosphoenol pyruvate-oxaloacetate-pyruvate node for POP-AA production, including L-tryptophan, L-tyrosine, L-phenylalanine, L-valine, L-lysine, L-threonine, and L-isoleucine. Novel heterologous routes and regulation methods regarding the carbon flux redistribution in the POP node and the formation of amino acids should be taken into consideration to improve POP-AA production to approach maximum theoretical values. Furthermore, an outlook for future strategies of low-cost feedstock and energy utilization for developing amino acid overproducers is proposed.
Assuntos
Aminoácidos , Engenharia Metabólica , Engenharia Metabólica/métodos , Aminoácidos/metabolismo , Ácido Oxaloacético/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Fosfoenolpiruvato/metabolismo , Corynebacterium glutamicum/metabolismo , Corynebacterium glutamicum/genética , Ácido Pirúvico/metabolismo , Redes e Vias Metabólicas , FermentaçãoRESUMO
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
Oxaloacetic acid (OAA) is a ß-ketocarboxylic acid, which plays an important role as an intermediate in some metabolic pathways, including the tricarboxylic acid cycle, gluconeogenesis and fatty acid biosynthesis. Animal studies have indicated that supplementing oxaloacetic acid shows an increase of lifespan and other substantial health benefits including mitochondrial DNA protection, and protection of retinal, neural and pancreatic tissues. Most of the chemical transformations of OAA in the metabolic pathways have been extensively studied; however, the understanding of decarboxylation of OAA at the atomic level is relatively lacking. Here, we carried out MD simulations and combined quantum mechanical/molecular mechanical (QM/MM) calculations as an example to systematically elucidate the binding modes, keto-enol tautomerization and decarboxylation of OAA in the active site of macrophomate synthase (MPS), which is a Mg(II)-dependent bifunctional enzyme that catalyzes both the decarboxylation of OAA and [4+2] cycloaddition of 2-pyrone with the decarboxylated intermediate of OAA (pyruvate enolate). On the basis of our calculations, it was found that the Mg2+-coordinated oxaloacetate may exist in enol forms and keto forms. The four keto forms can be transformed into each other by simply rotating the C2-C3 single bond, nevertheless, the keto-enol tautomerization strictly requires the assistance of pocket water molecules. In addition, the decarboxylation is stereo-electronically controlled, i.e., it is the relative orientation of the terminal carboxyl anion that determines the rate of decarboxylation. As such, the chemistry of oxaloacetate in the active site of MPS is complex. On one hand, the most stable binding mode (K-I) may undergo enol-keto tautomerization to isomerize to the enol form, which may further react with the second substrate; on the other hand, K-I may isomerize to another binding mode K-II to proceed decarboxylation to generate pyruvate enolate and CO2. Starting from K-I, the enol-keto tautomerization corresponds to a barrier of 16.2 kcal mol-1, whereas the decarboxylation is associated with an overall barrier of 19.7 kcal mol-1. These findings may provide useful information for understanding the chemistry of OAA and the catalysis of related enzymes, and they are basically in agreement with the available experimental kinetic data.
Assuntos
Ascomicetos , Complexos Multienzimáticos , Domínio Catalítico , Descarboxilação , Simulação de Dinâmica Molecular , Ácido Oxaloacético/metabolismo , Ácido Oxaloacético/química , Teoria Quântica , Estereoisomerismo , Complexos Multienzimáticos/química , Ascomicetos/enzimologiaRESUMO
BACKGROUND: Mitochondrial organelles play a crucial role in cellular metabolism so different cell types exhibit diverse metabolic and energy demands. Therefore, alternations in the intracellular distribution, quantity, function, and structure of mitochondria are required for stem cell differentiation. Finding an effective inducer capable of modulating mitochondrial activity is critical for the differentiation of specific stem cells into osteo-like cells for addressing issues related to osteogenic disorders. This study aimed to investigate the effect of oxaloacetate (OAA) on the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADSCs) in vitro. METHODS AND RESULTS: First, the most favorable OAA concentration was measured through MTT assay and subsequently confirmed using acridine orange staining. Human ADSCs were cultured in osteogenic medium supplemented with OAA and analyzed on days 7 and 14 of differentiation. Various assays including alkaline phosphatase assay (ALP), cellular calcium content assay, mineralized matrix staining with alizarin red, catalase (CAT) and superoxide dismutase (SOD) activity, and real-time RT-PCR analysis of three bone-specific markers (ALP, osteocalcin, and collagen type I) were conducted to characterize the differentiated cells. Following viability assessment, OAA at a concentration of 1 µM was considered the optimal dosage for further studies. The results of osteogenic differentiation assays showed that OAA at a concentration of 1 × 10- 6 M significantly increased ALP enzyme activity, mineralization, CAT and SOD activity and the expression of bone-specific genes in differentiated cells compared to control groups in vitro. CONCLUSIONS: In conclusion, the fundings from this study suggest that OAA possesses favorable properties that make it a potential candidate for application in medical bone regeneration.
Assuntos
Células-Tronco Mesenquimais , Osteogênese , Humanos , Tecido Adiposo/metabolismo , Ácido Oxaloacético/metabolismo , Células-Tronco Mesenquimais/metabolismo , Diferenciação Celular , Superóxido Dismutase/metabolismo , Células CultivadasRESUMO
The use of algae for industrial, biotechnological, and agricultural purposes is spreading globally. Scenedesmus species can play an essential role in the food industry and agriculture due to their favorable nutrient content and plant-stimulating properties. Previous research and the development of Scenedesmus-based foliar fertilizers raised several questions about the effectiveness of large-scale algal cultivation and the potential effects of algae on associative rhizobacteria. In the microbiological practice applied in agriculture, bacteria from the genus Azospirillum are one of the most studied plant growth-promoting, associative, nitrogen-fixing bacteria. Co-cultivation with Azospirillum species may be a new way of optimizing Scenedesmus culturing, but the functioning of the co-culture system still needs to be fully understood. It is known that Azospirillum brasilense can produce indole-3-acetic acid, which could stimulate algae growth as a plant hormone. However, the effect of microalgae on Azospirillum bacteria is unclear. In this study, we investigated the behavior of Azospirillum brasilense bacteria in the vicinity of Scenedesmus sp. or its supernatant using a microfluidic device consisting of physically separated but chemically coupled microchambers. Following the spatial distribution of bacteria within the device, we detected a positive chemotactic response toward the microalgae culture. To identify the metabolites responsible for this behavior, we tested the chemoeffector potential of citric acid and oxaloacetic acid, which, according to our HPLC analysis, were present in the algae supernatant in 0.074 mg/ml and 0.116 mg/ml concentrations, respectively. We found that oxaloacetic acid acts as a chemoattractant for Azospirillum brasilense.
Assuntos
Azospirillum brasilense , Scenedesmus , Scenedesmus/metabolismo , Microfluídica , Ácido Oxaloacético/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Plantas/metabolismoRESUMO
Fatigue is a common phenomenon closely related to physical discomfort and numerous diseases, which is severely threatening the life quality and health of people. However, the exact mechanisms underlying fatigue are not fully characterized. Herein, we demonstrate that oxaloacetic acid (OAA), a crucial tricarboxylic acid cycle intermediate, modulates the muscle fatigue. The results showed that serum OAA level was positively correlated with fatigue state of mice. OAA-treated induced muscle fatigue impaired the exercise performance of mice. Mechanistically, OAA increased the c-Jun N-terminal kinase (JNK) phosphorylation and uncoupling protein 2 (UCP2) levels in skeletal muscle, which led to decreased energy substrate and enhanced glycolysis. On the other hand, OAA boosted muscle mitochondrial oxidative phosphorylation uncoupled with energy production. In addition, either UCP2 knockout or JNK inhibition totally reversed the effects of OAA on skeletal muscle. Therein, JNK mediated UCP2 activation with OAA-treated. Our studies reveal a novel role of OAA in skeletal muscle metabolism, which would shed light on the mechanism of muscle fatigue and weakness.
Assuntos
Mitocôndrias , Ácido Oxaloacético , Humanos , Camundongos , Animais , Ácido Oxaloacético/metabolismo , Ácido Oxaloacético/farmacologia , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Ciclo do Ácido Cítrico , Músculo Esquelético/metabolismo , Proteína Desacopladora 2/genética , Proteína Desacopladora 2/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteína Desacopladora 3/metabolismo , Metabolismo EnergéticoRESUMO
A prevalent side-reaction of succinate dehydrogenase oxidizes malate to enol-oxaloacetate (OAA), a metabolically inactive form of OAA that is a strong inhibitor of succinate dehydrogenase. We purified from cow heart mitochondria an enzyme (OAT1) with OAA tautomerase (OAT) activity that converts enol-OAA to the physiological keto-OAA form, and determined that it belongs to the highly conserved and previously uncharacterized Fumarylacetoacetate_hydrolase_domain-containing protein family. From all three domains of life, heterologously expressed proteins were shown to have strong OAT activity, and ablating the OAT1 homolog caused significant growth defects. In Escherichia coli, expression of succinate dehydrogenase was necessary for OAT1-associated growth defects to occur, and ablating OAT1 caused a significant increase in acetate and other metabolites associated with anaerobic respiration. OAT1 increased the succinate dehydrogenase reaction rate by 35% in in vitro assays with physiological concentrations of both succinate and malate. Our results suggest that OAT1 is a universal metabolite repair enzyme that is required to maximize aerobic respiration efficiency by preventing succinate dehydrogenase inhibition.
Assuntos
Malatos , Succinato Desidrogenase , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , Malatos/metabolismo , Ciclo do Ácido Cítrico , Mitocôndrias Cardíacas/metabolismo , Oxaloacetatos/metabolismo , Ácido Oxaloacético/metabolismo , Malato Desidrogenase/metabolismoRESUMO
We previously found that skeletal muscle mitochondria incubated at low membrane potential (ΔΨ) or interscapular brown adipose tissue (IBAT) mitochondria, wherein ΔΨ is intrinsically low, accumulate oxaloacetate (OAA) in amounts sufficient to inhibit complex II respiration. We proposed a mechanism wherein low ΔΨ reduces reverse electron transport (RET) to complex I causing a low NADH/NAD+ ratio favoring malate conversion to OAA. To further assess the mechanism and its physiologic relevance, we carried out studies of mice with inherently different levels of IBAT mitochondrial inner membrane potential. Isolated complex II (succinate)-energized IBAT mitochondria from obesity-resistant 129SVE mice compared with obesity-prone C57BL/6J displayed greater UCP1 expression, similar O2 flux despite lower ΔΨ, similar OAA concentrations, and similar NADH/NAD+. When GDP was added to inhibit UCP1, 129SVE IBAT mitochondria, despite their lower ΔΨ, exhibited much lower respiration, twofold greater OAA concentrations, much lower RET (as marked by ROS), and much lower NADH and NADH/NAD+ ratios compared with the C57BL/6J IBAT mitochondria. UCP1 knock-out abolished OAA accumulation by succinate-energized mitochondria associated with markedly greater ΔΨ, ROS, and NADH, but equal or greater O2 flux compared with WT mitochondria. GDP addition, compared with no GDP, increased ΔΨ and complex II respiration in wild-type (WT) mice associated with much less OAA. Respiration on complex I substrates followed the more classical dynamics of greater respiration at lower ΔΨ. These findings support the abovementioned mechanism for OAA- and ΔΨ-dependent complex II respiration and support its physiological relevance.NEW & NOTEWORTHY We examined mitochondrial respiration initiated at mitochondrial complex II in mice with varying degrees of brown adipose tissue UCP1 expression. We show that, by affecting inner membrane potential, UCP1 expression determines reverse electron transport from complex II to complex I and, consequently, the NADH/NAD+ ratio. Accordingly, this regulates the level of oxaloacetate accumulation and the extent of oxaloacetate inhibition of complex II.
Assuntos
Tecido Adiposo Marrom , NAD , Camundongos , Animais , Tecido Adiposo Marrom/metabolismo , NAD/metabolismo , Ácido Oxaloacético/metabolismo , Ácido Oxaloacético/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Camundongos Endogâmicos C57BL , Respiração , Obesidade/metabolismo , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Potencial da Membrana Mitocondrial , Succinatos/metabolismo , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMO
At low inner mitochondrial membrane potential (ΔΨ) oxaloacetate (OAA) accumulates in the organelles concurrently with decreased complex II-energized respiration. This is consistent with ΔΨ-dependent OAA inhibition of succinate dehydrogenase. To assess the metabolic importance of this process, we tested the hypothesis that perturbing metabolic clearance of OAA in complex II-energized mitochondria would alter O2 flux and, further, that this would occur in both ΔΨ and tissue-dependent fashion. We carried out respiratory and metabolite studies in skeletal muscle and interscapular brown adipose tissue (IBAT) directed at the effect of OAA transamination to aspartate (catalyzed by the mitochondrial form of glutamic-oxaloacetic transaminase, Got2) on complex II-energized respiration. Addition of low amounts of glutamate to succinate-energized mitochondria at low ΔΨ increased complex II (succinate)-energized respiration in muscle but had little effect in IBAT mitochondria. The transaminase inhibitor, aminooxyacetic acid, increased OAA concentrations and impaired succinate-energized respiration in muscle but not IBAT mitochondria at low but not high ΔΨ. Immunoblotting revealed that Got2 expression was far greater in muscle than IBAT mitochondria. Because we incidentally observed metabolism of OAA to pyruvate in IBAT mitochondria, more so than in muscle mitochondria, we also examined the expression of mitochondrial oxaloacetate decarboxylase (ODX). ODX was detected only in IBAT mitochondria. In summary, at low but not high ΔΨ, mitochondrial transamination clears OAA preventing loss of complex II respiration: a process far more active in muscle than IBAT mitochondria. We also provide evidence that OAA decarboxylation clears OAA to pyruvate in IBAT mitochondria.
Assuntos
Ácido Oxaloacético , Succinato Desidrogenase , Ácido Oxaloacético/metabolismo , Succinato Desidrogenase/metabolismo , Tecido Adiposo Marrom , Músculo Esquelético/metabolismo , Respiração , Ácido Pirúvico/metabolismo , Ácido Succínico/metabolismoRESUMO
Malate dehydrogenase (MDH), which catalyzes a reversible conversion of L-malate to oxaloacetate, plays essential roles in common metabolic processes, such as the tricarboxylic acid cycle, the oxaloacetate-malate shuttle, and the glyoxylate cycle. MDH2 has lately been recognized as a promising anticancer target; however, the structural information for the human homologue with natural ligands is very limited. In this study, various complex structures of hMDH2, with its substrates and/or cofactors, were solved by X-ray crystallography, which could offer knowledge about the molecular and enzymatic mechanism of this enzyme and be utilized to design novel inhibitors. The structural comparison suggests that phosphate binds to the substrate binding site and brings the conformational change of the active loop to a closed state, which can secure the substate and cofactor to facilitate enzymatic activity.
Assuntos
Malato Desidrogenase , Malatos , Sítios de Ligação , Catálise , Cristalografia por Raios X , Glioxilatos , Humanos , Ligantes , Malato Desidrogenase/química , Malatos/química , NAD/metabolismo , Ácido Oxaloacético/química , Ácido Oxaloacético/metabolismo , FosfatosRESUMO
BACKGROUND: Many extracts and purified alkaloids of M. cordata (Papaveraceae family) have been reported to display promising anti-tumor effects by inhibiting cancer cell growth and inducing apoptosis in many cancer types. However, no evidence currently exists for anti-pancreatic cancer activity of alkaloids extracted from M. cordata, including a novel alkaloid named 6methoxy dihydrosphingosine (6-Methoxydihydroavicine, 6-ME) derived from M. cordata fruits. PURPOSE: The aim of this study was to investigate the anti-tumor effects of 6-ME on PC cells and the underlying mechanism. METHODS: CCK-8, RTCA, and colony-formation assays were used to analyze PC cell growth. Cell death ratios, changes in MMP and ROS levels were measured by flow cytometry within corresponding detection kits. A Seahorse XFe96 was employed to examine the effects of 6-ME on cellular bioenergetics. Western blot and q-RT-PCR were conducted to detect changes in target molecules. RESULTS: 6-ME effectively reduced the growth of PC cells and promoted PCD by activating RIPK1, caspases, and GSDME. Specifically, 6-ME treatment caused a disruption of OAA metabolism and increased ROS production, thereby affecting mitochondrial homeostasis and reducing aerobic glycolysis. These responses resulted in mitophagy and RIPK1-mediated cell death. CONCLUSION: 6-ME exhibited specific anti-tumor effects through interrupting OAA metabolic homeostasis to trigger ROS/RIPK1-dependent cell death and mitochondrial dysfunction, suggesting that 6-ME could be considered as a highly promising compound for PC intervention.
Assuntos
Alcaloides , Antineoplásicos , Caspases , Equol/análogos & derivados , Ácido Oxaloacético , Neoplasias Pancreáticas , Espécies Reativas de Oxigênio , Proteína Serina-Treonina Quinases de Interação com Receptores , Alcaloides/farmacologia , Antineoplásicos/farmacologia , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Equol/farmacologia , Humanos , Ácido Oxaloacético/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Papaveraceae/química , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
Oxidation of malate to oxaloacetate, catalyzed by either malate dehydrogenase (Mdh) or malate quinone oxidoreductase (Mqo), is a critical step of the tricarboxylic acid cycle. Both Mqo and Mdh are found in most bacterial genomes, but the level of functional redundancy between these enzymes remains unclear. A bioinformatic survey revealed that Mqo was not as widespread as Mdh in bacteria but that it was highly conserved in mycobacteria. We therefore used mycobacteria as a model genera to study the functional role(s) of Mqo and its redundancy with Mdh. We deleted mqo from the environmental saprophyte Mycobacterium smegmatis, which lacks Mdh, and found that Mqo was essential for growth on nonfermentable carbon sources. On fermentable carbon sources, the Δmqo mutant exhibited delayed growth and lowered oxygen consumption and secreted malate and fumarate as terminal end products. Furthermore, heterologous expression of Mdh from the pathogenic species Mycobacterium tuberculosis shortened the delayed growth on fermentable carbon sources and restored growth on nonfermentable carbon sources at a reduced growth rate. In M. tuberculosis, CRISPR interference of either mdh or mqo expression resulted in a slower growth rate compared to controls, which was further inhibited when both genes were knocked down simultaneously. These data reveal that exergonic Mqo activity powers mycobacterial growth under nonenergy limiting conditions and that endergonic Mdh activity complements Mqo activity, but at an energetic cost for mycobacterial growth. We propose Mdh is maintained in slow-growing mycobacterial pathogens for use under conditions such as hypoxia that require reductive tricarboxylic acid cycle activity.
Assuntos
Malato Desidrogenase , Malatos , Oxirredutases , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Ciclo do Ácido Cítrico , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Malatos/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Ácido Oxaloacético/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismoRESUMO
Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot, is a devastating necrotrophic pathogen which causes severe yield losses to oilseed production worldwide. Most of efforts at the genetic mitigation of the disease have not been successful. Present investigation was conducted to functionally characterize the effect of down-regulating Ssoah1 during host infection and explore the possibility of boosting host resistance by silencing this gene. We utilized host-induced gene silencing (HIGS) to silence Ssoah1 gene in the S. sclerotiorum fungus. A HIGS based vector was constructed and transformed into Arabidopsis thaliana. The pathogenicity assays in the transgenic A. thaliana lines revealed three T3 transformants with significantly higher resistance to S. sclerotiorum in comparison to untransformed controls. There was a concomitant reduction in expression of Ssoah1 and accumulation of oxalic acid in the necrotic regions of transgenic lines as compared to the non-transgenic controls. Specific Ssoah1-siRNA was highly expressed in HIGS Ssoah1 transgenic lines, as compared with WT and EV plants. The outcomes of oxalic acid estimation revealed that silencing of Ssoah1 results in decreased OA accumulation. The recovered mycelium plugs from HIGS Ssoah1 transgenic lines showed decreased Ssoah1 expression and pathogenesis. These results provide the possibility of using HIGS of Ssoah1 for engineering resistance against S. sclerotiorum.
Assuntos
Ascomicetos , Ácido Oxálico , Ascomicetos/metabolismo , Inativação Gênica , Ácido Oxálico/metabolismo , Ácido Oxaloacético/metabolismo , Doenças das Plantas/microbiologia , Virulência/genéticaRESUMO
Rabies, caused by rabies virus (RABV), is a widespread zoonosis that is nearly 100% fatal. Alteration of the metabolic environment affects viral replication and the immune response during viral infection. In this study, glucose uptake was increased in mouse brains at the late stage of infection with different RABV strains (lab-attenuated CVS strain and wild-type DRV strain). To illustrate the mechanism underlying glucose metabolism alteration, comprehensive analysis of lysine acetylation and target analysis of energy metabolites in mouse brains infected with CVS and DRV strains were performed. A total of 156 acetylated sites and 115 acetylated proteins were identified as significantly different during RABV infection. Compared to CVS- and mock-infected mice, the lysine acetylation levels of glycolysis and tricarboxylic acid (TCA) cycle enzymes were decreased, and enzyme activity was upregulated in DRV-infected mouse brains. Metabolomic analysis revealed high levels of oxaloacetate (OAA) in RABV-infected mouse brains. Specifically, the OAA level in CVS-infected mouse brains was higher than that in DRV-infected mouse brains, which contributed to the enhancement of the metabolic rate at the substrate level. Finally, we confirmed that OAA could reduce excessive neuroinflammation in CVS-infected mouse brains by inhibiting JNK and P38 phosphorylation. Taken together, this study provides fresh insight into the different strategies the host adapts to regulate glucose metabolism for energy requirements after different RABV strain infections and suggests that OAA treatment is a strategy to prevent neural damage during RABV infection. IMPORTANCE Both viral replication and the host immune response are highly energy dependent. It is important to understand how the rabies virus affects energy metabolism in the brain. Glucose is the direct energy source for cell metabolism. Previous studies have revealed that there is some association between acetylation and metabolic processes. In this study, comprehensive protein acetylation and glucose metabolism analysis were conducted to compare glucose metabolism in mouse brains infected with different RABV strains. Our study demonstrates that the regulation of enzyme activity by acetylation and OAA accumulation at the substrate level are two strategies for the host to respond to energy requirements after RABV infection. Our study also indicates the role OAA could play in neuronal protection by suppressing excessive neuroinflammation.
Assuntos
Encéfalo/metabolismo , Glucose/metabolismo , Vírus da Raiva/patogenicidade , Raiva/metabolismo , Acetilação , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/imunologia , Encéfalo/virologia , Metabolismo Energético , Inflamação , Camundongos , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Ácido Oxaloacético/metabolismo , Ácido Oxaloacético/uso terapêutico , Proteoma/metabolismo , Raiva/tratamento farmacológico , Raiva/virologiaRESUMO
Glutamate plays diverse roles in neuronal cells, affecting cell energetics and reactive oxygen species (ROS) generation. These roles are especially vital for neuronal cells, which deal with high amounts of glutamate as a neurotransmitter. Our analysis explored neuronal glutamate implication in cellular energy metabolism and ROS generation, using a kinetic model that simulates electron transport details in respiratory complexes, linked ROS generation and metabolic reactions. The analysis focused on the fact that glutamate attenuates complex II inhibition by oxaloacetate, stimulating the latter's transformation into aspartate. Such a mechanism of complex II activation by glutamate could cause almost complete reduction of ubiquinone and deficiency of oxidized form (Q), which closes the main stream of electron transport and opens a way to massive ROS generating transfer in complex III from semiquinone radicals to molecular oxygen. In this way, under low workload, glutamate triggers the respiratory chain (RC) into a different steady state characterized by high ROS generation rate. The observed stepwise dependence of ROS generation on glutamate concentration experimentally validated this prediction. However, glutamate's attenuation of oxaloacetate's inhibition accelerates electron transport under high workload. Glutamate-oxaloacetate interaction in complex II regulation underlies the observed effects of uncouplers and inhibitors and acceleration of Ca2+ uptake. Thus, this theoretical analysis uncovered the previously unknown roles of oxaloacetate as a regulator of ROS generation and glutamate as a modifier of this regulation. The model predicted that this mechanism of complex II activation by glutamate might be operative in situ and responsible for excitotoxicity. Spatial-time gradients of synthesized hydrogen peroxide concentration, calculated in the reaction-diffusion model with convection under a non-uniform local approximation of nervous tissue, have shown that overproduction of H2O2 in a cell causes excess of its level in neighbor cells.
Assuntos
Encéfalo/metabolismo , Ácido Glutâmico/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Ácido Oxaloacético/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sinapses/metabolismo , Trifosfato de Adenosina/metabolismo , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Transporte Biológico/efeitos dos fármacos , Cálcio/metabolismo , Respiração Celular/efeitos dos fármacos , Complexo II de Transporte de Elétrons/metabolismo , Metabolismo Energético/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Cinética , Metacrilatos/farmacologia , Mitocôndrias/efeitos dos fármacos , Imagens de Fantasmas , Sinapses/efeitos dos fármacos , Tiazóis/farmacologia , Fatores de TempoRESUMO
Pharmacological activation of the glycolytic enzyme PKM2 or expression of the constitutively active PKM1 isoform in cancer cells results in decreased lactate production, a phenomenon known as the PKM2 paradox in the Warburg effect. Here we show that oxaloacetate (OAA) is a competitive inhibitor of human lactate dehydrogenase A (LDHA) and that elevated PKM2 activity increases de novo synthesis of OAA through glutaminolysis, thereby inhibiting LDHA in cancer cells. We also show that replacement of human LDHA with rabbit LDHA, which is relatively resistant to OAA inhibition, eliminated the paradoxical correlation between the elevated PKM2 activity and the decreased lactate concentration in cancer cells treated with a PKM2 activator. Furthermore, rabbit LDHA-expressing tumours, compared to human LDHA-expressing tumours in mice, displayed resistance to the PKM2 activator. These findings describe a mechanistic explanation for the PKM2 paradox by showing that OAA accumulates and inhibits LDHA following PKM2 activation.
Assuntos
Ácido Oxaloacético/metabolismo , Piruvato Quinase/metabolismo , Animais , Linhagem Celular Tumoral , Citosol/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Glucose/metabolismo , Glicólise , Humanos , Lactato Desidrogenase 5/antagonistas & inibidores , Lactato Desidrogenase 5/metabolismo , Camundongos , Piruvato Quinase/genética , CoelhosRESUMO
In this contribution the dissociative electron attachment to metabolites found in aerobic organisms, namely oxaloacetic and citric acids, was studied both experimentally by means of a crossed-beam setup and theoretically through density functional theory calculations. Prominent negative ion resonances from both compounds are observed peaking below 0.5 eV resulting in intense formation of fragment anions associated with a decomposition of the carboxyl groups. In addition, resonances at higher energies (3-9 eV) are observed exclusively from the decomposition of the oxaloacetic acid. These fragments are generated with considerably smaller intensities. The striking findings of our calculations indicate the different mechanism by which the near 0 eV electron is trapped by the precursor molecule to form the transitory negative ion prior to dissociation. For the oxaloacetic acid, the transitory anion arises from the capture of the electron directly into some valence states, while, for the citric acid, dipole- or multipole-bound states mediate the transition into the valence states. What is also of high importance is that both compounds while undergoing DEA reactions generate highly reactive neutral species that can lead to severe cell damage in a biological environment.
Assuntos
Ânions/química , Ácido Cítrico/química , Ácido Oxaloacético/química , Ânions/metabolismo , Ácido Cítrico/metabolismo , Elétrons , Gases/química , Modelos Teóricos , Ácido Oxaloacético/metabolismo , Teoria QuânticaRESUMO
At the junction between the glycolysis and the tricarboxylic acid cycle-as well as various other metabolic pathways-lies the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node (PPO-node). These three metabolites form the core of a network involving at least eleven different types of enzymes, each with numerous subtypes. Obviously, no single organism maintains each of these eleven enzymes; instead, different organisms possess different subsets in their PPO-node, which results in a remarkable degree of variation, despite connecting such deeply conserved metabolic pathways as the glycolysis and the tricarboxylic acid cycle. The PPO-node enzymes play a crucial role in cellular energetics, with most of them involved in (de)phosphorylation of nucleotide phosphates, while those responsible for malate conversion are important redox enzymes. Variations in PPO-node therefore reflect the different energetic niches that organisms can occupy. In this review, we give an overview of the biochemistry of these eleven PPO-node enzymes. We attempt to highlight the variation that exists, both in PPO-node compositions, as well as in the roles that the enzymes can have within those different settings, through various recent discoveries in both bacteria and archaea that reveal deviations from canonical functions.
Assuntos
Metabolismo Energético , Enzimas/metabolismo , Ácido Oxaloacético/metabolismo , Fosfoenolpiruvato/metabolismo , Ácido Pirúvico/metabolismo , Archaea/enzimologia , Bactérias/enzimologiaRESUMO
The tricarboxylic acid (TCA) cycle is one of the most important metabolic pathways in nature. Oxygenic photoautotrophic bacteria, cyanobacteria, have an unusual TCA cycle. The TCA cycle in cyanobacteria contains two unique enzymes that are not part of the TCA cycle in other organisms. In recent years, sustainable metabolite production from carbon dioxide using cyanobacteria has been looked at as a means to reduce the environmental burden of this gas. Among cyanobacteria, the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) is an optimal host for sustainable metabolite production. Recently, metabolite production using the TCA cycle in Synechocystis 6803 has been carried out. Previous studies revealed that the branch point of the oxidative and reductive TCA cycles, oxaloacetate metabolism, plays a key role in metabolite production. However, the biochemical mechanisms regulating oxaloacetate metabolism in Synechocystis 6803 are poorly understood. Concentrations of oxaloacetate in Synechocystis 6803 are extremely low, such that in vivo analysis of oxaloacetate metabolism does not seem realistic. Therefore, using purified enzymes, we reconstituted oxaloacetate metabolism in Synechocystis 6803 in vitro to reveal the regulatory mechanisms involved. Reconstitution of oxaloacetate metabolism revealed that pH, Mg2+ and phosphoenolpyruvate are important factors affecting the conversion of oxaloacetate in the TCA cycle. Biochemical analyses of the enzymes involved in oxaloacetate metabolism in this and previous studies revealed the biochemical mechanisms underlying the effects of these factors on oxaloacetate conversion. In addition, we clarified the function of two l-malate dehydrogenase isozymes in oxaloacetate metabolism. These findings serve as a basis for various applications of the cyanobacterial TCA cycle.
Assuntos
Ciclo do Ácido Cítrico , Ácido Oxaloacético/metabolismo , Synechocystis/metabolismo , Fumaratos/metabolismo , Concentração de Íons de Hidrogênio , Cloreto de Magnésio/metabolismo , Malato Desidrogenase/metabolismo , Fosfoenolpiruvato/metabolismo , TemperaturaRESUMO
Isotope-based assessment of metabolic flux is achieved through a judicious balance of measurements and assumptions. Recent publications debate the validity of key assumptions used to model stable isotope labeling of liver metabolism in vivo. Here, we examine the controversy surrounding estimates of liver citric acid cycle and gluconeogenesis fluxes using a flexible modeling platform that enables rigorous testing of standard assumptions. Fasted C57BL/6J mice are infused with [13C3]lactate or [13C3]propionate isotopes, and hepatic fluxes are regressed using models with gradually increasing complexity and relaxed assumptions. We confirm that liver pyruvate cycling fluxes are incongruent between different 13C tracers in models with conventional assumptions. When models are expanded to include more labeling measurements and fewer constraining assumptions, however, liver pyruvate cycling is significant, and inconsistencies in hepatic flux estimates using [13C3]lactate and [13C3]propionate isotopes emanate, in part, from peripheral tracer recycling and incomplete isotope equilibration within the citric acid cycle.