RESUMEN
The demand for sustainably produced bulk chemicals is constantly rising. Succinate serves as a fundamental component in various food, chemical, and pharmaceutical products. Succinate can be produced from sustainable raw materials using microbial fermentation and enzyme-based technologies. Bacteroides and Phocaeicola species, widely distributed and prevalent gut commensals, possess enzyme sets for the metabolization of complex plant polysaccharides and synthesize succinate as a fermentative end product. This study employed novel molecular techniques to enhance succinate yields in the natural succinate producer Phocaeicola vulgatus by directing the metabolic carbon flow toward succinate formation. The deletion of the gene encoding the methylmalonyl-CoA mutase (Δmcm, bvu_0309-0310) resulted in a 95% increase in succinate production, as metabolization to propionate was effectively blocked. Furthermore, deletion of genes encoding the lactate dehydrogenase (Δldh, bvu_2499) and the pyruvate:formate lyase (Δpfl, bvu_2880) eliminated the formation of fermentative end products lactate and formate. By overproducing the transketolase (TKT, BVU_2318) in the triple deletion mutant, succinate production increased from 3.9 mmol/g dry weight in the wild type to 10.9 mmol/g dry weight. Overall, succinate yield increased by 180% in the new mutant strain P. vulgatus Δmcm Δldh Δpfl pG106_tkt relative to the parent strain. This approach is a proof of concept, verifying the genetic accessibility of P. vulgatus, and forms the basis for targeted genetic optimization. The increase of efficiency highlights the huge potential of P. vulgatus as a succinate producer with applications in sustainable bioproduction processes. KEY POINTS: ⢠Deleting methylmalonyl-CoA mutase gene in P. vulgatus doubled succinate production ⢠Triple deletion mutant with transketolase overexpression increased succinate yield by 180% ⢠P. vulgatus shows high potential for sustainable bulk chemical production via genetic optimization.
Asunto(s)
Fermentación , Ácido Succínico , Ácido Succínico/metabolismo , Humanos , Ingeniería Metabólica/métodos , Eliminación de Gen , Metilmalonil-CoA Mutasa/genética , Metilmalonil-CoA Mutasa/metabolismo , Microbioma Gastrointestinal , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Succinic acid (SA), a dicarboxylic acid of industrial importance, can be efficiently produced by metabolically engineered Mannheimia succiniciproducens. Although the importance of magnesium (Mg2+) ion on SA production has been evident from our previous studies, the role of Mg2+ ion remains largely unexplored. In this study, we investigated the impact of Mg2+ ion on SA production and developed a hyper-SA producing strain of M. succiniciproducens by reconstructing the Mg2+ ion transport system. To achieve this, optimal alkaline neutralizer comprising Mg2+ ion was developed and the physiological effect of Mg2+ ion was analyzed. Subsequently, the Mg2+ ion transport system was reconstructed by introducing an efficient Mg2+ ion transporter from Salmonella enterica. A high-inoculum fed-batch fermentation of the final engineered strain produced 152.23 ± 0.99 g/L of SA, with a maximum productivity of 39.64 ± 0.69 g/L/h. These findings highlight the importance of Mg2+ ions and transportation system optimization in succinic acid production by M. succiniciproducens.
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Fermentación , Magnesio , Mannheimia , Ácido Succínico , Ácido Succínico/metabolismo , Magnesio/metabolismo , Mannheimia/metabolismo , Mannheimia/genética , Ingeniería Metabólica/métodos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas de Transporte de Catión/metabolismo , Proteínas de Transporte de Catión/genéticaRESUMEN
Effect of succinic acid on the processes of myogenesis was investigated in the study with the cells of C2C12 line. In the concentration range 10-1000 µM, succinic acid stimulated the process of myogenic differentiation, increasing the levels of myogenesis factors MyoD (at all stages of myogenesis) and myogenin (at the stage of terminal differentiation). Presence of the succinate receptors SUCNR1 was revealed in the C2C12 cells using Western blotting, level of which decreased during myogenesis. When succinic acid was added to the cells, the level of intracellular succinate did not change significantly and decreased during myogenic differentiation. Using a specific Gai protein inhibitor, pertussis toxin, it was found that stimulation of myogenesis in the C2C12 cells under the action of succinic acid is realized through SUCNR1-Gai interaction.
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Diferenciación Celular , Desarrollo de Músculos , Ácido Succínico , Ácido Succínico/metabolismo , Desarrollo de Músculos/efectos de los fármacos , Animales , Ratones , Diferenciación Celular/efectos de los fármacos , Línea Celular , Receptores Acoplados a Proteínas G/metabolismo , Proteína MioD/metabolismo , Miogenina/metabolismoRESUMEN
BACKGROUND: Cartilage metabolism dysregulation is a crucial driver in knee osteoarthritis (KOA). Modulating the homeostasis can mitigate the cartilage degeneration in KOA. Curcumenol, derived from traditional Chinese medicine Curcuma Longa L., has demonstrated potential in enhancing chondrocyte proliferation and reducing apoptosis. However, the specific mechanism of Curcumenol in treating KOA remains unclear. This study aimed to demonstrate the molecular mechanism of Curcumenol in treating KOA based on the transcriptomics and metabolomics, and both in vivo and in vitro experimental validations. MATERIALS AND METHODS: In this study, a destabilization medial meniscus (DMM)-induced KOA mouse model was established. And the mice were intraperitoneally injected with Curcumenol at 4 and 8 mg/kg concentrations. The effects of Curcumenol on KOA cartilage and subchondral was evaluated using micro-CT, histopathology, and immunohistochemistry (IHC). In vitro, OA chondrocytes were induced with 10 µg/mL lipopolysaccharide (LPS) and treated with Curcumenol to evaluate the proliferation, apoptosis, and extracellular matrix (ECM) metabolism through CCK8 assay, flow cytometry, and chondrocyte staining. Furthermore, transcriptomics and metabolomics were utilized to identify differentially expressed genes (DEGs) and metabolites. Finally, integrating multi-omics analysis, virtual molecular docking (VMD), and molecular dynamics simulation (MDS), IHC, immunofluorescence (IF), PCR, and Western blot (WB) validation were conducted to elucidate the mechanism by which Curcumenol ameliorates KOA cartilage degeneration. RESULTS: Curcumenol ameliorated cartilage destruction and subchondral bone loss in KOA mice, promoted cartilage repair, upregulated the expression of COL2 while downregulated MMP3, and improved ECM synthesis metabolism. Additionally, Curcumenol also alleviated the damage of LPS on the proliferation activity and suppressed apoptosis, promoted ECM synthesis. Transcriptomic analysis combined with weighted gene co-expression network analysis (WGCNA) identified a significant downregulation of 19 key genes in KOA. Metabolomic profiling showed that Curcumenol downregulates the expression of d-Alanyl-d-alanine, 17a-Estradiol, Glutathione, and Succinic acid, while upregulating Sterculic acid and Azelaic acid. The integrated multi-omics analysis suggested that Curcumenol targeted KDM6B to regulate downstream protein H3K27me3 expression, which inhibited methylation at the histone H3K27, consequently reducing Succinic acid levels and improving KOA cartilage metabolism homeostasis. Finally, both in vivo and in vitro findings indicated that Curcumenol upregulated KDM6B, suppressed H3K27me3 expression, and stimulated collagen II expression and ECM synthesis, thus maintaining cartilage metabolism homeostasis and alleviating KOA cartilage degeneration. CONCLUSION: Curcumenol promotes cartilage repair and ameliorates cartilage degeneration in KOA by upregulating KDM6B expression, thereby reducing H3K27 methylation and downregulating Succinic Acid, restoring metabolic stability and ECM synthesis.
Asunto(s)
Condrocitos , Curcuma , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Osteoartritis de la Rodilla , Ácido Succínico , Animales , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Ratones , Masculino , Curcuma/química , Osteoartritis de la Rodilla/tratamiento farmacológico , Osteoartritis de la Rodilla/metabolismo , Ácido Succínico/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Proliferación Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Sesquiterpenos/farmacología , Simulación del Acoplamiento Molecular , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/efectos de los fármacos , HumanosRESUMEN
Mortierella alpina is popular for lipid production, but the low carbon conversion rate and lipid yield are major obstacles for its economic performance. Here, external addition of organic acids involved in tricarboxylic acid cycle was used to tune carbon flux and improve lipid production. Citrate was determined to be the best organic acid that can be used for enhancing lipid production. By the addition of citrate, the lipid titer and content were approximately 1.24 and 1.34 times higher, respectively. Meanwhile, citrate supplement also promoted the accumulation of succinate, an important value-added platform chemical. Owing to the improved lipid and succinate production through adding citrate, the carbon conversion rate of M. alpina reached up to 52.17%, much higher than that of the control group (14.11%). The addition of citrate could redistribute carbon flux by regulating the expression level of genes related to tricarboxylic acid cycle metabolism. More carbon fluxes flow to lipid and succinate synthesis, which greatly improved the carbon conversion efficiency of M. alpina. This study provides an effective and straightforward strategy with potential economic benefits to improve carbon conversion efficiency in M. alpina.
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Carbono , Ciclo del Ácido Cítrico , Ácido Cítrico , Mortierella , Ácido Succínico , Mortierella/metabolismo , Mortierella/genética , Ácido Succínico/metabolismo , Carbono/metabolismo , Ácido Cítrico/metabolismo , Lípidos/biosíntesis , Metabolismo de los Lípidos , Regulación Fúngica de la Expresión Génica , FermentaciónRESUMEN
BACKGROUND: Studies have shown that m6A modification is related to the occurrence and development of papillary thyroid carcinoma (PTC). The disorder of succinic acid metabolism is associated with the occurrence and development of various tumors. However, there are few studies based on m6A and succinate metabolism-related genes (SMRGs) in PTC. METHODS: The TCGA-Thyroid carcinoma (THCA), GSE33630, 1159 SMRGs, and 23 m6A regulatory factors were collected from the online databases. Subsequently, the differentially expressed genes (DEGs) were selected between PTC (Tumor) and Normal samples. The overlapping genes among the DEGs, m6A, and SMRGs were applied to screen the biomarkers. Using the 3 machine-learning algorithms, the biomarkers were determined based on the overlapping genes. Next, the biomarkers were evaluated by the ROC curve and expression analysis in TCGA-THCA and GSE33630. Then, the overall survival (OS) differences were compared between the high-and low-expression biomarkers. Finally, immune infiltration analysis, molecular regulatory network, and drug prediction were performed based on the biomarkers. RESULTS: In TCGA-THCA, there were 2800 DEGs between and Normal samples, and then 7 overlapping genes were obtained. Importantly, ADK, TNFRSF10B, CYP7B1, FGFR2, and CPQ were determined as biomarkers with excellent diagnostic efficiency (AUC > 0.7). In PTC samples, ADK and TNFRSF10B were high-expressed while CYP7B1, FGFR2, and CPQ were low-expressed. Especially, the high-expression groups of ADK had a better prognosis, while the high-expression groups of CYP7B1, FGFR2, and CPQ had a worse prognosis. Afterward, immune infiltration analysis found that 16 immune cells had infiltration differences between the Tumor and Normal samples. Finally, transcription factor SP1 could regulate CYP7B1 and TNFRSF10B. Moreover, Navitoclax was a potential drug for PTC patients. CONCLUSION: Overall, we described 5 biomarkers associated with adverse prognosis of PTC, including ADK, TNFRSF10B, CYP7B1, FGFR2, and CPQ. All these biomarkers were involved in succinate metabolism and m6A modification of RNA. This set of biomarkers should be explored further for their diagnostic value in PTC. Investigations into the mechanistic role of alteration of succinate metabolism and m6A modification of RNA pathways in the pathophysiology of PTC are warranted.
Asunto(s)
Biomarcadores de Tumor , Ácido Succínico , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides , Humanos , Cáncer Papilar Tiroideo/genética , Cáncer Papilar Tiroideo/metabolismo , Cáncer Papilar Tiroideo/patología , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Ácido Succínico/metabolismo , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Pronóstico , Perfilación de la Expresión Génica , Adenosina/análogos & derivadosRESUMEN
Succinate was found extensive applications in the food additives, pharmaceutical, and biopolymers industries. However, the succinate biosynthesis in E. coli required IPTG, lacked NADH, and produced high yields only under anaerobic conditions, unsuitable for cell growth. To overcome these limitations, the glyoxylate shunt and reductive TCA pathway were simultaneously enhanced to produce succinate in both aerobic and anaerobic conditions and achieve a high cell growth meanwhile. On this basis, NADH availability and sugars uptake were increased. Furthermore, an oxygen-dependent promoter was used to dynamically regulate the expression level of key genes of reductive TCA pathway to avoid the usage of IPTG. The final strain E. coli Mgls7-32 could produce succinate from corn stover hydrolysate without an inducer, achieving a titer of 72.8 g/L in 5 L bioreactor (1.2 mol/mol of total sugars). Those findings will aid in the industrial production of succinate.
Asunto(s)
Ciclo del Ácido Cítrico , Escherichia coli , Fermentación , Glioxilatos , Ácido Succínico , Zea mays , Ácido Succínico/metabolismo , Escherichia coli/metabolismo , Glioxilatos/metabolismo , Hidrólisis , Oxidación-Reducción , Reactores Biológicos , AnaerobiosisRESUMEN
The gut microbiome plays an important role in honeybee hormonal regulation and growth, but the underlying mechanisms are poorly understood. Here, we showed that the depletion of gut bacteria resulted in reduced expression of insulin-like peptide gene (ilp) in the head, accompanied by metabolic syndromes resembling those of Type 1 diabetes in humans: hyperglycemia, impaired lipid storage, and decreased metabolism. These symptoms were alleviated by gut bacterial inoculation. Gut metabolite profiling revealed that succinate, produced by Lactobacillus Firm-5, played deterministic roles in activating ilp gene expression and in regulating metabolism in honeybees. Notably, we demonstrated that succinate modulates host ilp gene expression through stimulating gut gluconeogenesis, a mechanism resembling that of humans. This study presents evidence for the role of gut metabolite in modulating host metabolism and contributes to the understanding of the interactions between gut microbiome and bee hosts.
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Microbioma Gastrointestinal , Lactobacillus , Ácido Succínico , Abejas/microbiología , Animales , Microbioma Gastrointestinal/efectos de los fármacos , Lactobacillus/metabolismo , Ácido Succínico/metabolismo , GluconeogénesisRESUMEN
Cancerous genetic mutations result in a complex and comprehensive post-translational modification (PTM) dynamics, in which protein succinylation is well known for its ability to reprogram cell metabolism and is involved in the malignant evolution. Little is known about the regulatory interactions between succinylation and other PTMs in the PTM network. Here, we developed a conjoint analysis and systematic clustering method to explore the intermodification communications between succinylome and phosphorylome from eight lung cancer patients. We found that the intermodification coorperation in both parallel and series. Besides directly participating in metabolism pathways, some phosphosites out of mitochondria were identified as an upstream regulatory modification directing succinylome dynamics in cancer metabolism reprogramming. Phosphorylated activation of histone deacetylase (HDAC) in lung cancer resulted in the removal of acetylation and favored the occurrence of succinylation modification of mitochondrial proteins. These results suggest a tandem regulation between succinylation and phosphorylation in the PTM network and provide HDAC-related targets for intervening mitochondrial succinylation and cancer metabolism reprogramming.
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Histona Desacetilasas , Neoplasias Pulmonares , Procesamiento Proteico-Postraduccional , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Fosforilación , Histona Desacetilasas/metabolismo , Ácido Succínico/metabolismo , Mitocondrias/metabolismoRESUMEN
Succinic acid is an important C4 platform compound that serves as a raw material for the production of 1,4-butanediol, tetrahydrofuran, and biodegradable plastics such as polybutylene succinate (PBS). Compared to the traditional petrochemical-based route that uses maleic anhydride as a raw material, the microbial fermentation method for producing succinic acid offers more sustainable economic value and environmental friendliness. Yeasts with good acid tolerance can achieve low-pH fermentation of succinic acid, significantly reducing the cost of product extraction. Therefore, constructing high-yield succinic acid yeast strains through metabolic engineering has garnered increasing attention. This paper systematically introduced the application value and market size of succinic acid, summarized the pathways and key enzymes involved in succinic acid synthesis in microorganisms, and elaborated on the latest research progress in the synthesis of succinic acid using yeast cell factories. It also presented the current status of succinic acid synthesis using non-food raw materials such as glycerol, acetic acid, lignocellulosic hydrolysate, and others as substrates by engineered yeast strains. Finally, the paper provided a prospect for low-pH succinic acid biomanufacturing based on yeast cell factories.
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Fermentación , Ingeniería Metabólica , Saccharomyces cerevisiae , Ácido Succínico , Ácido Succínico/metabolismo , Ingeniería Metabólica/métodos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Microbiología Industrial , Butileno Glicoles/metabolismoRESUMEN
Succinic acid (SA) is a valuable C4 platform chemical with diverse applications. Lignocellulosic biomass represents an abundant and renewable carbon resource for microbial production of SA. However, the presence of toxic compounds in pretreated lignocellulosic hydrolysates poses challenges to cell metabolism, leading to inefficient SA production. Here, engineered Yarrowia lipolytica Hi-SA2 was shown to utilize glucose and xylose from corncob hydrolysate to produce 32.6 g/L SA in shaking flasks. The high concentration of undetoxified hydrolysates significantly inhibited yeast growth and SA biosynthesis, with furfural identified as the key inhibitor. Through overexpressing glutathione synthetase encoding gene YlGsh2, the tolerance of engineered strain to furfural and toxic hydrolysate was significantly improved. In a 5-L bioreactor, Hi-SA2-YlGsh2 strain produced 45.34 g/L SA within 32 h, with a final pH of 3.28. This study provides a sustainable process for bio-based SA production, highlighting the efficient SA synthesis from lignocellulosic biomass through low pH fermentation.
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Fermentación , Lignina , Ácido Succínico , Yarrowia , Yarrowia/metabolismo , Yarrowia/genética , Lignina/metabolismo , Ácido Succínico/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Reactores Biológicos , Biomasa , Glucosa/metabolismo , Xilosa/metabolismo , Ingeniería Metabólica/métodos , Ingeniería Genética/métodos , Furaldehído/metabolismoRESUMEN
OBJECTIVES: Syntrophy has been documented between pectinophiles and methanol-utilizing bacteria, along with instances of cross-feeding between pectinophiles and methanogens. However, studies on the ecology of pectinophiles in anaerobic digestion (AD) are lacking. Therefore, in this study, we aimed to elucidate the ecology of pectinophiles by isolating novel pectinophile forms and conducting a comprehensive analysis of their physiology and ecology. METHODS: Complex microbial communities from AD systems were enriched in a pectin-containing medium; subsequently, specific strains were isolated using a pectinophile isolation method. The carbon source assimilation and growth ability of the isolates, along with their symbiotic relationships, were evaluated using batch tests. RESULTS: Strain LPYR103-Pre exhibited 16S rRNA gene sequence similarity and average nucleotide identity values of 94.3 % and 77.9 %, respectively, compared to its closest related species, Segatella cerevisiae. Strain LPYR103-Pre demonstrated attenuated growth in the presence of eight common sugars but exhibited remarkably high growth in the presence of pectin, d-galacturonate, and d-glucuronate, with succinate being identified as a primary metabolite. Accumulation of succinate inhibited the growth of strain LPYR103-Pre. However, this growth impediment was alleviated by Dialister hominis LPYG114-Dih, whose growth required succinate. CONCLUSIONS: Our results elucidate the specific carbon source requirements of the Segatella-like strain LPYR103-Pre and succinate-mediated symbiosis involving D. hominis. These findings provide new insights into the degradation of pectin and its degradation products during AD, contributing to the identification of unknown pectinophiles.
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Pectinas , Filogenia , ARN Ribosómico 16S , Ácido Succínico , Simbiosis , ARN Ribosómico 16S/genética , Ácido Succínico/metabolismo , Pectinas/metabolismo , AnaerobiosisRESUMEN
Lonicera japonica plays a significant role in traditional Chinese medicine and as a food source, making it a focus of studies on protein succinylation and its potential role in regulating secondary metabolism during flower development. This study aimed to clarify the regulatory mechanism of protein succinylation on phenylpropanoid-related phenotypic changes by conducting a global lysine succinylation proteomic analysis across different flowering stages. A total of 586 lysine succinylated peptides in 303 proteins were identified during early and late floral stages. Functional enrichment analysis revealed that succinylated proteins primarily participated in the tricarboxylic acid (TCA) cycle, amino acid metabolism, and secondary metabolism. The abundance of succinylated aspartate transaminase (AT), 4-coumarate-CoA ligase (4CL), and phenylalanine N-hydroxylase (CYP79A2) in phenylpropanoid metabolism varied during flower development. In vitro experiments demonstrated that succinylation increased AT activity while inhibited 4CL activity. Decreased levels of total flavonoids and phenolic acids indicated significant alterations in phenylpropanoid metabolism during later floral stages. These results suggest that succinylation of TCA cycle proteins not only influences flower development but also, together with AT-4CL-CYP79A2 co-succinylation, redirects phenylpropanoid metabolism during flower development in L. japonica.
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Flores , Lonicera , Lisina , Proteínas de Plantas , Flores/metabolismo , Flores/crecimiento & desarrollo , Proteínas de Plantas/metabolismo , Lisina/metabolismo , Lonicera/metabolismo , Lonicera/crecimiento & desarrollo , Procesamiento Proteico-Postraduccional , Ácido Succínico/metabolismo , Proteómica/métodosRESUMEN
BACKGROUND: Biotransformation of CO2 into high-value-added carbon-based products is a promising process for reducing greenhouse gas emissions. To realize the green transformation of CO2, we use fatty acids as carbon source to drive CO2 fixation to produce succinate through a portion of the 3-hydroxypropionate (3HP) cycle in Cupriavidus necator H16. RESULTS: This work can achieve the production of a single succinate molecule from one acetyl-CoA molecule and two CO2 molecules. It was verified using an isotope labeling experiment utilizing NaH13CO3. This implies that 50% of the carbon atoms present in succinate are derived from CO2, resulting in a twofold increase in efficiency compared to prior methods of succinate biosynthesis that relied on the carboxylation of phosphoenolpyruvate or pyruvate. Meanwhile, using fatty acid as a carbon source has a higher theoretical yield than other feedstocks and also avoids carbon loss during acetyl-CoA and succinate production. To further optimize succinate production, different approaches including the optimization of ATP and NADPH supply, optimization of metabolic burden, and optimization of carbon sources were used. The resulting strain was capable of producing succinate to a level of 3.6 g/L, an increase of 159% from the starting strain. CONCLUSIONS: This investigation established a new method for the production of succinate by the implementation of two CO2 fixation reactions and demonstrated the feasibility of ATP, NADPH, and metabolic burden regulation strategies in biological carbon fixation.
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Dióxido de Carbono , Cupriavidus necator , Ácidos Grasos , Ácido Succínico , Dióxido de Carbono/metabolismo , Cupriavidus necator/metabolismo , Ácidos Grasos/metabolismo , Ácido Succínico/metabolismo , Acetilcoenzima A/metabolismo , NADP/metabolismoRESUMEN
Tobacco smoking is the main etiological factor of lung cancer (LC), which can also cause metabolome disruption. This study aimed to investigate whether the observed metabolic shift in LC patients was also associated with their smoking status. Untargeted metabolomics profiling was applied for the initial screening of changes in serum metabolic profile between LC and chronic obstructive pulmonary disease (COPD) patients, selected as a non-cancer group. Differences in metabolite profiles between current and former smokers were also tested. Then, targeted metabolomics methods were applied to verify and validate the proposed LC biomarkers. For untargeted metabolomics, a single extraction-dual separation workflow was applied. The samples were analyzed using a liquid chromatograph-high resolution quadrupole time-of-flight mass spectrometer. Next, the selected metabolites were quantified using liquid chromatography-triple-quadrupole mass spectrometry. The acquired data confirmed that patients' stratification based on smoking status impacted the discriminating ability of the identified LC marker candidates. Analyzing a validation set of samples enabled us to determine if the putative LC markers were truly robust. It demonstrated significant differences in the case of four metabolites: allantoin, glutamic acid, succinic acid, and sphingosine-1-phosphate. Our research showed that studying the influence of strong environmental factors, such as tobacco smoking, should be considered in cancer marker research since it reduces the risk of false positives and improves understanding of the metabolite shifts in cancer patients.
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Biomarcadores de Tumor , Neoplasias Pulmonares , Metabolómica , Fumar , Humanos , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/metabolismo , Metabolómica/métodos , Biomarcadores de Tumor/sangre , Masculino , Femenino , Persona de Mediana Edad , Fumar/sangre , Fumar/efectos adversos , Anciano , Esfingosina/análogos & derivados , Esfingosina/sangre , Esfingosina/metabolismo , Lisofosfolípidos/sangre , Lisofosfolípidos/metabolismo , Metaboloma , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/sangre , Cromatografía Liquida/métodos , Ácido Succínico/sangre , Ácido Succínico/metabolismo , Ácido Glutámico/sangre , Ácido Glutámico/metabolismoRESUMEN
Organic acid metabolites exhibit acidic properties. These metabolites serve as intermediates in major carbon metabolic pathways and are involved in several biochemical pathways, including the tricarboxylic acid (TCA) cycle and glycolysis. They also regulate cellular activity and play crucial roles in epigenetics, tumorigenesis, and cellular signal transduction. Knowledge of the binding proteins of organic acid metabolites is crucial for understanding their biological functions. However, identifying the binding proteins of these metabolites has long been a challenging task owing to the transient and weak nature of their interactions. Moreover, traditional methods are unsuitable for the structural modification of the ligands of organic acid metabolites because these metabolites have simple and similar structures. Even minor structural modifications can significantly affect protein interactions. Thermal proteome profiling (TPP) provides a promising avenue for identifying binding proteins without the need for structural modifications. This approach has been successfully applied to the identification of the binding proteins of several metabolites. In this study, we investigated the binding proteins of two TCA cycle intermediates, i.e., succinate and fumarate, and lactate, an end-product of glycolysis, using the matrix thermal shift assay (mTSA) technique. This technique involves combining single-temperature (52 â) TPP and dose-response curve analysis to identify ligand-binding proteins with high levels of confidence and determine the binding affinity between ligands and proteins. To this end, HeLa cells were lysed, followed by protein desalting to remove endogenous metabolites from the cell lysates. The desalted cell lysates were treated with fumarate or succinate at final concentrations of 0.004, 0.04, 0.4, and 2 mmol/L in the experimental groups or 2 mmol/L sodium chloride in the control group. Considering that the cellular concentration of lactate can be as high as 2-30 mmol/L, we then applied lactate at final concentrations of 0.2, 1, 5, 10, and 25 mmol/L in the experimental groups or 25 mmol/L sodium chloride in the control group. Using high-sensitivity mass spectrometry coupled with data-independent acquisition (DIA) quantification, we quantified 5870, 5744, and 5816 proteins in succinate, fumarate, and lactate mTSA experiments, respectively. By setting stringent cut-off values (i.e., significance of changes in protein thermal stability (p-value)<0.001 and quality of the dose-response curve fitting (square of Pearson's correlation coefficient, R2)>0.95), multiple binding proteins for these organic acid metabolites from background proteins were confidently determined. Several known binding proteins were identified, notably fumarate hydratase (FH) as a binding protein for fumarate, and α-ketoglutarate-dependent dioxygenase (FTO) as a binding protein for both fumarate and succinate. Additionally, the affinity data for the interactions between these metabolites and their binding proteins were obtained, which closely matched those reported in the literature. Interestingly, ornithine aminotransferase (OAT), which is involved in amino acid biosynthesis, and 3-mercaptopyruvate sulfurtransferase (MPST), which acts as an antioxidant in cells, were identified as lactate-binding proteins. Subsequently, an orthogonal assay technique developed in our laboratory, the solvent-induced precipitation (SIP) technique, was used to validate the mTSA results. SIP identified OAT as the top target candidate, validating the mTSA-based finding that OAT is a novel lactate-binding protein. Although MPST was not identified as a lactate-binding protein by SIP, statistical analysis of MPST in the mTSA experiments with 10 or 25 mmol/L lactate revealed that MPST is a lactate-binding protein with a high level of confidence. Peptide-level empirical Bayes t-tests combined with Fisher's exact test also supported the conclusion that MPST is a lactate-binding protein. Lactate is structurally similar to pyruvate, the known binding protein of MPST. Therefore, assuming that lactate could potentially occupy the binding site of pyruvate on MPST. Overall, the novel binding proteins identified for lactate suggest their potential involvement in amino acid synthesis and redox balance regulation.
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Ciclo del Ácido Cítrico , Humanos , Células HeLa , Ácido Succínico/metabolismo , Ácido Succínico/química , Fumaratos/metabolismo , Fumaratos/químicaRESUMEN
Abnormal polarization of adipose tissue macrophages (ATMs) results in low-grade systemic inflammation and insulin resistance (IR), potentially contributing to the development of diabetes. However, the underlying mechanisms that regulate the polarization of ATMs associated with gestational diabetes mellitus (GDM) remain unclear. Thus, we aimed to determine the effects of abnormal fatty acids on macrophage polarization and development of insulin resistance in GDM. Levels of fatty acids and inflammation were assessed in the serum samples and adipose tissues of patients with GDM. An in vitro cell model treated with palmitic acid was established, and the mechanisms of palmitic acid in regulating macrophage polarization was clarified. The effects of excessive palmitic acid on the regulation of histone methylations and IR were also explored in the high-fat diet induced GDM mice model. We found that pregnancies with GDM were associated with increased levels of serum fatty acids, and inflammation and IR in adipose tissues. Increased palmitic acid could induce mitochondrial dysfunction and excessive ROS levels in macrophages, leading to abnormal cytoplasmic and nuclear metabolism of succinate and α-ketoglutarate (αKG). Specifically, a decreased nuclear αKG/succinate ratio could attenuate the enrichment of H3K27me3 at the promoters of pro-inflammatory cytokines, such as IL-1ß, IL-6, and TNF-α, leading to cytokine secretion. Importantly, GDM mice treated with GSK-J4, an inhibitor of histone lysine demethylase, were protected from abnormal pro-inflammatory macrophage polarization and excessive production of pro-inflammatory cytokines. Our findings highlight the importance of the metabolism of αKG and succinate as transcriptional modulators in regulating the polarization of ATMs and the insulin sensitivity of adipose tissue, ensuring a normal pregnancy. This novel insight sheds new light on gestational fatty acid metabolism and epigenetic alterations associated with GDM.
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Tejido Adiposo , Diabetes Gestacional , Resistencia a la Insulina , Ácidos Cetoglutáricos , Macrófagos , Ácido Palmítico , Ácido Succínico , Diabetes Gestacional/metabolismo , Diabetes Gestacional/patología , Embarazo , Ácido Palmítico/farmacología , Animales , Femenino , Ratones , Tejido Adiposo/metabolismo , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/patología , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Humanos , Ácidos Cetoglutáricos/metabolismo , Ácido Succínico/metabolismo , Dieta Alta en Grasa/efectos adversos , Adulto , Ratones Endogámicos C57BL , Inflamación/metabolismo , Inflamación/patología , Modelos Animales de EnfermedadRESUMEN
We studied the respiratory activity of mitochondria in peripheral blood leukocytes from 36 patients with coronary heart disease (CHD) and a history of ventricular tachyarrhythmias required cardioverter-defibrillator implantation. The measurements were carried out in incubation buffers with different oxidation substrates (succinate and pyruvate-malate mixture). In pyruvate-malate incubation buffer, oxygen consumption rate and respiratory control coefficients in patients with triggered device did not differ significantly from those in patients without cardioverter-defibrillator triggering. At the same time, respiratory control coefficients were below the reference values. In succinate buffer, values of mitochondrial parameters were significantly lower in patients with triggered devices. Our findings indicate that mitochondria of patients with non-triggered cardioverters-defibrillators have better functional and metabolic plasticity. It was concluded that activity of respiratory processes in mitochondria could be an indicator that should be taken into the account when assessing the risk of developing ventricular tachyarrhythmias.
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Enfermedad Coronaria , Desfibriladores Implantables , Consumo de Oxígeno , Humanos , Masculino , Persona de Mediana Edad , Enfermedad Coronaria/fisiopatología , Enfermedad Coronaria/terapia , Consumo de Oxígeno/fisiología , Femenino , Mitocondrias/metabolismo , Anciano , Taquicardia Ventricular/fisiopatología , Taquicardia Ventricular/terapia , Ácido Pirúvico/metabolismo , Ácido Succínico/metabolismo , Malatos/metabolismo , Mitocondrias Cardíacas/metabolismoRESUMEN
Microbial interactions impact the functioning of microbial communities. However, microbial interactions within host-associated communities remain poorly understood. Here, we report that the beneficiary rhizobacterium Niallia sp. RD1 requires the helper Pseudomonas putida H3 for bacterial growth and beneficial interactions with the plant host. In the absence of the helper H3 strain, the Niallia sp. RD1 strain exhibited weak respiration and elongated cell morphology without forming bacterial colonies. A transposon mutant of H3 in a gene encoding succinate-semialdehyde dehydrogenase displayed much attenuated support of RD1 colony formation. Through the subsequent addition of succinate to the media, we found that succinate serves as a public good that supports RD1 growth. Comparative genome analysis highlighted that RD1 lacked the gene for sufficient succinate, suggesting its evolution as a beneficiary of succinate biosynthesis. The syntrophic interaction between RD1 and H3 efficiently protected tomato plants from bacterial wilt and promoted tomato growth. The addition of succinate to the medium restored complex II-dependent respiration in RD1 and facilitated the cultivation of various bacterial isolates from the rhizosphere. Taken together, we delineate energy auxotrophic beneficiaries ubiquitous in the microbial community, and these beneficiaries could benefit host plants with the aid of helpers in the rhizosphere.
Asunto(s)
Rizosfera , Solanum lycopersicum , Ácido Succínico , Solanum lycopersicum/microbiología , Ácido Succínico/metabolismo , Interacciones Microbianas , Microbiología del Suelo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Pseudomonas putida/crecimiento & desarrolloRESUMEN
Metabolic reprogramming and energetic rewiring are hallmarks of cancer that fuel disease progression and facilitate therapy evasion. The remodelling of oxidative phosphorylation and enhanced lipogenesis have previously been characterised as key metabolic features of prostate cancer (PCa). Recently, succinate-dependent mitochondrial reprogramming was identified in high-grade prostate tumours, as well as upregulation of the enzymes associated with branched-chain amino acid (BCAA) catabolism. In this study, we hypothesised that the degradation of the BCAAs, particularly valine, may play a critical role in anapleurotic refuelling of the mitochondrial succinate pool, as well as the maintenance of intracellular lipid metabolism. Through the suppression of BCAA availability, we report significantly reduced lipid content, strongly indicating that BCAAs are important lipogenic fuels in PCa. This work also uncovered a novel compensatory mechanism, whereby fatty acid uptake is increased in response to extracellular valine deprivation. Inhibition of valine degradation via suppression of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) resulted in a selective reduction of malignant prostate cell proliferation, decreased intracellular succinate and impaired cellular respiration. In combination with a comprehensive multi-omic investigation that incorporates next-generation sequencing, metabolomics, and high-content quantitative single-cell imaging, our work highlights a novel therapeutic target for selective inhibition of metabolic reprogramming in PCa.