ABSTRACT
BACKGROUND: Helicobacter pylori (H. pylori) infection is critical in the development and occurrence of gastric cancer. H. pylori secretes gamma-glutamyl transferase (GGT), which affects energy metabolism and histone methylation in mesenchymal stem cells. However, its effect on human gastric epithelial cells remains unclear. This study aimed to investigate the effects of GGT on energy metabolism and histone methylation in gastric epithelial cells and determine its role in the development and progression of H. pylori-induced gastric cancer. METHODS: A GGT knockout H. pylori strain and mouse gastric cancer model were constructed, and alpha-ketoglutarate (α-KG) was added. The underlying mechanism was investigated using proteomics, immunohistochemistry, Western blotting, and other experimental assays. RESULTS: H. pylori can colonize the host's stomach and destroy the gastric epithelium. GGT secreted by H. pylori decreased the concentration of glutamine in the stomach and increased H3K9me3 and H3K27me3 expression, which promoted the proliferation and migration of gastric epithelial cells. Additionally, α-KG reversed this effect. GGT increased the tumorigenic ability of nude mice. GGT, secreted by H. pylori, promoted the expression of ribosomal protein L15 (RPL15), while GGT knockout and supplementation with α-KG and trimethylation inhibitors reduced RPL15 expression and Wnt signaling pathway expression. CONCLUSIONS: H. pylori secreted GGT decreased the expression of glutamine and α-KG in gastric epithelial cells, increased the expression of histones H3K9me3 and H3K27me3, and activated the Wnt signaling pathway through RPL15 expression, ultimately changing the biological characteristics of the gastric epithelium and promoting the occurrence of gastric cancer. Altered energy metabolism and histone hypermethylation are important factors involved in this process.
Subject(s)
Energy Metabolism , Epithelial Cells , Helicobacter pylori , Histones , Stomach Neoplasms , gamma-Glutamyltransferase , Stomach Neoplasms/microbiology , Stomach Neoplasms/pathology , Stomach Neoplasms/metabolism , Stomach Neoplasms/genetics , Animals , Histones/metabolism , Methylation , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Epithelial Cells/pathology , gamma-Glutamyltransferase/metabolism , gamma-Glutamyltransferase/genetics , Mice , Humans , Mice, Nude , Gastric Mucosa/metabolism , Gastric Mucosa/microbiology , Gastric Mucosa/pathology , Cell Proliferation , Helicobacter Infections/metabolism , Helicobacter Infections/microbiology , Helicobacter Infections/pathology , Helicobacter Infections/complications , Ketoglutaric Acids/metabolismABSTRACT
Alpha-ketoglutarate (α-KG), an endogenous intermediate of the tricarboxylic acid cycle, is involved in a variety of cellular metabolic pathways. It serves as an energy donor, a precursor of amino acid biosynthesis, and an epigenetic regulator. α-KG plays physiological functions in immune regulation, oxidative stress, and anti-aging as well. In recent years, it has been reported that the level of α-KG in the body is closely associated with metabolic syndrome, including obesity, hyperglycemia, and other pathological factors. Exogenous supplementation of α-KG improves obesity, blood glucose levels, and cardiovascular disease risks associated with metabolic syndrome. Furthermore, α-KG regulates the common pathological mechanisms of metabolic syndrome, suggesting the potential application prospect of α-KG in metabolic syndrome. In order to provide a theoretical basis for further exploration of the application of α-KG in metabolic syndrome, we focused on α-KG and metabolic syndrome in this article and summarized the latest research progress in the role of α-KG in improving the pathological condition and disease progression of metabolic syndrome. For the next step, researchers may focus on the co-pathogenesis of metabolic syndrome and investigate whether α-KG can be used to achieve the therapeutic goal of "homotherapy for heteropathy" in the treatment of metabolic syndrome.
Subject(s)
Ketoglutaric Acids , Metabolic Syndrome , Metabolic Syndrome/metabolism , Ketoglutaric Acids/metabolism , Humans , Obesity/metabolism , Obesity/complications , Animals , Oxidative StressABSTRACT
The incidence of colorectal cancer (CRC) is closely linked to metabolic diseases. Accumulating evidence suggests the regulatory role of AMP-activated protein kinase (AMPK) in cancer metabolic reprogramming. In this study, wild-type and AMPK knockout mice were subjected to azoxymethane-induced and dextran sulfate sodium (AOM/DSS)-promoted colitis-associated CRC induction. A stable AMPK-deficient Caco-2 cell line was also established for the mechanistic studies. The data showed that AMPK deficiency accelerated CRC development, characterized by increased tumor number, tumor size, and hyperplasia in AOM/DSS-treated mice. The aggravated colorectal tumorigenesis resulting from AMPK ablation was associated with reduced α-ketoglutarate production and ten-eleven translocation hydroxylase 2 (TET2) transcription, correlated with the reduced mismatch repair protein mutL homolog 1 (MLH1) protein. Furthermore, in AMPK-deficient Caco-2 cells, the mRNA expression of mismatch repair and tumor suppressor genes, intracellular α-ketoglutarate, and the protein level of TET2 were also downregulated. AMPK deficiency also increased hypermethylation in the CpG islands of Mlh1 in both colonic tissues and Caco-2 cells. In conclusion, AMPK deficiency leads to reduced α-ketoglutarate concentration and elevates the suppressive epigenetic modifications of tumor suppressor genes in gut epithelial cells, thereby increasing the risk of colorectal tumorigenesis. Given the modifiable nature of AMPK activity, it holds promise as a prospective molecular target for the prevention and treatment of CRC.
Subject(s)
AMP-Activated Protein Kinases , Azoxymethane , Carcinogenesis , Colorectal Neoplasms , DNA Methylation , Dioxygenases , Animals , Humans , Mice , AMP-Activated Protein Kinases/genetics , AMP-Activated Protein Kinases/metabolism , Azoxymethane/toxicity , Azoxymethane/adverse effects , Caco-2 Cells , Carcinogenesis/genetics , Colitis/chemically induced , Colitis/genetics , Colitis/pathology , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Colorectal Neoplasms/chemically induced , Colorectal Neoplasms/etiology , Dextran Sulfate/toxicity , Dioxygenases/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic , Ketoglutaric Acids/metabolism , Mice, Knockout , MutL Protein Homolog 1/genetics , MutL Protein Homolog 1/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolismABSTRACT
Osteoarthritis (OA) is the most common degenerative joint disorder that causes disability in aged individuals, caused by functional and structural alterations of the knee joint. To investigate whether metabolic drivers might be harnessed to promote cartilage repair, a liquid chromatography-mass spectrometry (LC-MS) untargeted metabolomics approach was carried out to screen serum biomarkers in osteoarthritic rats. Based on the correlation analyses, α-ketoglutarate (α-KG) has been demonstrated to have antioxidant and anti-inflammatory properties in various diseases. These properties make α-KG a prime candidate for further investigation of OA. Experimental results indicate that α-KG significantly inhibited H2O2-induced cartilage cell matrix degradation and apoptosis, reduced levels of reactive oxygen species (ROS) and malondialdehyde (MDA), increased superoxide dismutase (SOD) and glutathione (GSH)/glutathione disulfide (GSSG) levels, and upregulated the expression of ETV4, SLC7A11 and GPX4. Further mechanistic studies observed that α-KG, like Ferrostatin-1 (Fer-1), effectively alleviated Erastin-induced apoptosis and ECM degradation. α-KG and Fer-1 upregulated ETV4, SLC7A11, and GPX4 at the mRNA and protein levels, decreased ferrous ion (Fe2+) accumulation, and preserved mitochondrial membrane potential (MMP) in ATDC5 cells. In vivo, α-KG treatment inhibited ferroptosis in OA rats by activating the ETV4/SLC7A11/GPX4 pathway. Thus, these findings indicate that α-KG inhibits ferroptosis via the ETV4/SLC7A11/GPX4 signaling pathway, thereby alleviating OA. These observations suggest that α-KG exhibits potential therapeutic properties for the treatment and prevention of OA, thereby having potential clinical applications in the future.
Subject(s)
Ferroptosis , Ketoglutaric Acids , Osteoarthritis , Phospholipid Hydroperoxide Glutathione Peroxidase , Signal Transduction , Ferroptosis/drug effects , Animals , Osteoarthritis/drug therapy , Osteoarthritis/metabolism , Osteoarthritis/pathology , Ketoglutaric Acids/metabolism , Ketoglutaric Acids/pharmacology , Signal Transduction/drug effects , Rats , Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism , Phospholipid Hydroperoxide Glutathione Peroxidase/genetics , Amino Acid Transport System y+/metabolism , Amino Acid Transport System y+/genetics , Male , Proto-Oncogene Proteins c-ets/metabolism , Proto-Oncogene Proteins c-ets/genetics , Rats, Sprague-Dawley , Apoptosis/drug effects , Reactive Oxygen Species/metabolismABSTRACT
Severe bleeding from deep and irregular wounds poses a significant challenge in prehospital and surgical settings. To address this issue, we developed a novel chitosan-based hemostatic dressing with a magnetic targeting mechanism using Fe3O4, termed bovine serum albumin-modified Fe3O4 embedded in porous α-ketoglutaric acid/chitosan (BSA/Fe3O4@KA/CS). This dressing enhances hemostasis by magnetically guiding the agent to the wound site. In vitro, the hemostatic efficacy of BSA/Fe3O4@KA/CS is comparable to that of commercial chitosan (Celox™) and is not diminished by the modification. In vivo, BSA/Fe3O4@KA/CS demonstrated superior hemostatic performance and reduced blood loss compared to Celox™. The hemostatic mechanism of BSA/Fe3O4@KA/CS includes the concentration of solid blood components through water absorption, adherence to blood cells, and activation of the endogenous coagulation pathway. Magnetic field targeting is crucial in directing the dressing to deep hemorrhagic sites. Additionally, safety assessments have confirmed the biocompatibility and biodegradability of BSA/Fe3O4@KA/CS. In conclusion, we introduce a novel approach to modify chitosan using magnetic guidance for effective hemostasis, positioning BSA/Fe3O4@KA/CS as a promising candidate for managing various wounds.
Subject(s)
Bandages , Chitosan , Hemostatics , Serum Albumin, Bovine , Chitosan/chemistry , Serum Albumin, Bovine/chemistry , Animals , Hemostatics/chemistry , Hemostatics/pharmacology , Porosity , Ketoglutaric Acids/chemistry , Ketoglutaric Acids/pharmacology , Cattle , Male , Hemorrhage/drug therapy , Hemorrhage/therapy , MiceABSTRACT
N-Acetylnorloline synthase (LolO) is one of several iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases that catalyze sequential reactions of different types in the biosynthesis of valuable natural products. LolO hydroxylates C2 of 1-exo-acetamidopyrrolizidine before coupling the C2-bonded oxygen to C7 to form the tricyclic loline core. Each reaction requires cleavage of a C-H bond by an oxoiron(IV) (ferryl) intermediate; however, different carbons are targeted, and the carbon radicals have different fates. Prior studies indicated that the substrate-cofactor disposition (SCD) controls the site of H· abstraction and can affect the reaction outcome. These indications led us to determine whether a change in SCD from the first to the second LolO reaction might contribute to the observed reactivity switch. Whereas the single ferryl complex in the C2 hydroxylation reaction was previously shown to have typical Mössbauer parameters, one of two ferryl complexes to accumulate during the oxacyclization reaction has the highest isomer shift seen to date for such a complex and abstracts H· from C7 â¼ 20 times faster than does the first ferryl complex in its previously reported off-pathway hydroxylation of C7. The detectable hydroxylation of C7 in competition with cyclization by the second ferryl complex is not enhanced in 2H2O solvent, suggesting that the C2 hydroxyl is deprotonated prior to C7-H cleavage. These observations are consistent with the coordination of the C2 oxygen to the ferryl complex, which may reorient its oxo ligand, the substrate, or both to positions more favorable for C7-H cleavage and oxacyclization.
Subject(s)
Iron , Ketoglutaric Acids , Ketoglutaric Acids/metabolism , Ketoglutaric Acids/chemistry , Iron/metabolism , Iron/chemistry , Hydroxylation , Cyclization , Oxygenases/metabolism , Oxygenases/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/chemistryABSTRACT
α-Ketoglutarate (AKG), a crucial intermediate in the tricarboxylic acid cycle, has been demonstrated to mitigate hyperlipidemia-induced dyslipidemia and endothelial damage. While hyperlipidemia stands as a major trigger for non-alcoholic fatty liver disease, the protection of AKG on hyperlipidemia-induced hepatic metabolic disorders remains underexplored. This study aims to investigate the potential protective effects and mechanisms of AKG against hepatic lipid metabolic disorders caused by acute hyperlipidemia. Our observations indicate that AKG effectively alleviates hepatic lipid accumulation, mitochondrial dysfunction, and loss of redox homeostasis in P407-induced hyperlipidemia mice, as well as in palmitate-injured HepG2 cells and primary hepatocytes. Mechanistic insights reveal that the preventive effects are mediated by activating the AMPK-PGC-1α/Nrf2 pathway. In conclusion, our findings shed light on the role and mechanism of AKG in ameliorating abnormal lipid metabolic disorders in hyperlipidemia-induced fatty liver, suggesting that AKG, an endogenous mitochondrial nutrient, holds promising potential for addressing hyperlipidemia-induced fatty liver conditions.
Subject(s)
AMP-Activated Protein Kinases , Hyperlipidemias , Ketoglutaric Acids , NF-E2-Related Factor 2 , Oxidative Stress , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Signal Transduction , Animals , Hyperlipidemias/metabolism , Hyperlipidemias/drug therapy , Hyperlipidemias/complications , Mice , Oxidative Stress/drug effects , Humans , NF-E2-Related Factor 2/metabolism , AMP-Activated Protein Kinases/metabolism , Ketoglutaric Acids/metabolism , Ketoglutaric Acids/pharmacology , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/genetics , Signal Transduction/drug effects , Hep G2 Cells , Mitochondria/metabolism , Mitochondria/drug effects , Male , Lipid Metabolism/drug effects , Hepatocytes/metabolism , Hepatocytes/drug effects , Fatty Liver/metabolism , Fatty Liver/etiology , Fatty Liver/drug therapy , Fatty Liver/prevention & control , Fatty Liver/pathology , Disease Models, Animal , Liver/metabolism , Liver/drug effects , Liver/pathologyABSTRACT
OBJECTIVE: In this pilot study, we used untargeted metabolomics to identify biochemical mechanisms or biomarkers potentially underlying SLE-related fatigue. METHODS: Metabolon conducted untargeted metabolomic plasma profiling using ultrahigh performance liquid chromatography/tandem mass spectrometry on plasma samples of 23 Black females with systemic lupus erythematosus (SLE) and 21 no SLE controls. Fatigue phenotypes of general fatigue, physical fatigue, mental fatigue, reduced activity, and reduced motivation were measured with the reliable and valid Multidimensional Fatigue Inventory (MFI). RESULTS: A total of 290 metabolites were significantly different between the SLE and no SLE groups, encompassing metabolites related to glycolysis, TCA cycle activity, heme catabolism, branched chain amino acids, fatty acid metabolism, and steroids. Within the SLE group, controlling for age and co-morbidities, TCA cycle metabolites of alpha-ketoglutarate (AKG) and succinate were statistically significantly associated (p < .05) with physical and general fatigue. CONCLUSION: While pervasive perturbations in the entire TCA cycle have been implicated as a potential mechanism for fatigue, our results suggest individual metabolites of AKG and succinate may be potential biomarkers or targets of intervention for fatigue symptom management in SLE. Additionally, perturbations in heme metabolism in the SLE group provide additional insights into mechanisms that promote systemic inflammation.
Subject(s)
Biomarkers , Citric Acid Cycle , Fatigue , Lupus Erythematosus, Systemic , Metabolomics , Humans , Female , Lupus Erythematosus, Systemic/blood , Lupus Erythematosus, Systemic/complications , Lupus Erythematosus, Systemic/physiopathology , Pilot Projects , Fatigue/etiology , Fatigue/blood , Adult , Metabolomics/methods , Biomarkers/blood , Middle Aged , Black or African American , Tandem Mass Spectrometry , Case-Control Studies , Succinic Acid/blood , Ketoglutaric Acids/blood , Chromatography, High Pressure LiquidABSTRACT
Enzymes of the central metabolism tend to assemble into transient supramolecular complexes. However, the functional significance of the interactions, particularly between enzymes catalyzing non-consecutive reactions, remains unclear. Here, by co-localizing two non-consecutive enzymes of the TCA cycle from Bacillus subtilis, malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICD), in phase separated droplets we show that MDH-ICD interaction leads to enzyme agglomeration with a concomitant enhancement of ICD catalytic rate and an apparent sequestration of its reaction product, 2-oxoglutarate. Theory demonstrates that MDH-mediated clustering of ICD molecules explains the observed phenomena. In vivo analyses reveal that MDH overexpression leads to accumulation of 2-oxoglutarate and reduction of fluxes flowing through both the catabolic and anabolic branches of the carbon-nitrogen intersection occupied by 2-oxoglutarate, resulting in impeded ammonium assimilation and reduced biomass production. Our findings suggest that the MDH-ICD interaction is an important coordinator of carbon-nitrogen metabolism.
Subject(s)
Bacillus subtilis , Carbon , Citric Acid Cycle , Isocitrate Dehydrogenase , Ketoglutaric Acids , Malate Dehydrogenase , Nitrogen , Nitrogen/metabolism , Carbon/metabolism , Malate Dehydrogenase/metabolism , Malate Dehydrogenase/genetics , Bacillus subtilis/metabolism , Bacillus subtilis/genetics , Bacillus subtilis/enzymology , Isocitrate Dehydrogenase/metabolism , Isocitrate Dehydrogenase/genetics , Ketoglutaric Acids/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Ammonium Compounds/metabolismABSTRACT
Extracellular matrix (ECM) is a major component of the tumor environment, promoting the establishment of a pro-invasive behavior. Such environment is supported by both tumor- and stromal-derived metabolites, particularly lactate. In prostate cancer (PCa), cancer-associated fibroblasts (CAFs) are major contributors of secreted lactate, able to impact on metabolic and transcriptional regulation in cancer cells. Here, we describe a mechanism by which CAF-secreted lactate promotes in PCa cells the expression of genes coding for the collagen family. Lactate-exploiting PCa cells rely on increased α-ketoglutarate (α-KG) which activates the α-KG-dependent collagen prolyl-4-hydroxylase (P4HA1) to support collagen hydroxylation. De novo synthetized collagen plays a signaling role by activating discoidin domain receptor 1 (DDR1), supporting stem-like and invasive features of PCa cells. Inhibition of lactate-induced collagen hydroxylation and DDR1 activation reduces the metastatic colonization of PCa cells. Overall, these results provide a new understanding of the link between collagen remodeling/signaling and the nutrient environment exploited by PCa.
Subject(s)
Collagen , Extracellular Matrix , Lactic Acid , Neoplasm Metastasis , Prostatic Neoplasms , Male , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Prostatic Neoplasms/genetics , Humans , Extracellular Matrix/metabolism , Collagen/metabolism , Lactic Acid/metabolism , Cell Line, Tumor , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Signal Transduction , Gene Expression Regulation, Neoplastic , Animals , Mice , Tumor Microenvironment , Ketoglutaric Acids/metabolism , HydroxylationABSTRACT
The fat mass and obesity-associated fatso (FTO) protein is a member of the Alkb family of dioxygenases and catalyzes oxidative demethylation of N6-methyladenosine (m6A), N1-methyladenosine (m1A), 3-methylthymine (m3T), and 3-methyluracil (m3U) in single-stranded nucleic acids. It is well established that the catalytic activity of FTO proceeds via two coupled reactions. The first reaction involves decarboxylation of alpha-ketoglutarate (αKG) and formation of an oxyferryl species. In the second reaction, the oxyferryl intermediate oxidizes the methylated nucleic acid to reestablish Fe(II) and the canonical base. However, it remains unclear how binding of the nucleic acid activates the αKG decarboxylation reaction and why FTO demethylates different methyl modifications at different rates. Here, we investigate the interaction of FTO with 5-mer DNA oligos incorporating the m6A, m1A, or m3T modifications using solution NMR, molecular dynamics (MD) simulations, and enzymatic assays. We show that binding of the nucleic acid to FTO activates a two-state conformational equilibrium in the αKG cosubstrate that modulates the O2 accessibility of the Fe(II) catalyst. Notably, the substrates that provide better stabilization to the αKG conformation in which Fe(II) is exposed to O2 are demethylated more efficiently by FTO. These results indicate that i) binding of the methylated nucleic acid is required to expose the catalytic metal to O2 and activate the αKG decarboxylation reaction, and ii) the measured turnover of the demethylation reaction (which is an ensemble average over the entire sample) depends on the ability of the methylated base to favor the Fe(II) state accessible to O2.
Subject(s)
Alpha-Ketoglutarate-Dependent Dioxygenase FTO , Iron , Ketoglutaric Acids , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolism , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/chemistry , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Ketoglutaric Acids/metabolism , Ketoglutaric Acids/chemistry , Iron/metabolism , Iron/chemistry , Humans , Substrate Specificity , Adenosine/analogs & derivatives , Adenosine/metabolism , Adenosine/chemistry , Protein Conformation , Uracil/metabolism , Uracil/analogs & derivatives , Uracil/chemistry , Molecular Dynamics Simulation , Thymine/analogs & derivativesABSTRACT
Total triterpenoids from the fruits of Chaenomeles speciosa(TCS) are active components in the prevention and treatment of gastric mucosal damage, which have potential anti-aging effects. However, it is still unclear whether TCS can improve gastric aging, especially its molecular mechanism against gastric aging. On this basis, this study explored the effect and mechanism of TCS on senescent GES-1 cells induced by D-galactose(D-gal) to provide scientific data for the clinical use of TCS to prevent gastric aging. GES-1 cells cultured in vitro and those transfected with overexpression GLS1(GLS1-OE) plasmid of glutaminase 1(GLS1) were induced to aging by D-gal, and then TCS and or GLS1 inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide(BPTES) were given. Cell survival rate, positive rate of ß-galactosidase(SA-ß-gal) staining, mitochondrial membrane potential(MMP), and apoptosis were investigated. GLS1 activity, levels of glutamine(Gln), glutamate(Glu), α-ketoglutarate(α-KG), urea, and ammonia in supernatant and cells were detected by enzyme-linked immunosorbent assay(ELISA) and colorimetric methods. The mRNA and protein expressions of GLS1 and the related genes of the mitochondrial apoptosis signaling pathway were measured by real-time fluorescence quantitative PCR and Western blot. The results manifested that compared with the D-gal model group and GLS1-OE D-gal model group, TCS significantly decreased the SA-ß-gal staining positive cell rate and MMP of D-gal-induced senescent GES-1 cells and GLS1-OE senescent GES-1 cells, inhibited the survival of senescent cells, and promoted their apoptosis(P<0.01). It decreased the activity of GLS1 and the content of Gln, Glu, α-KG, urea, and ammonia in supernatant and cell(P<0.01), reduced the concentration of cytochrome C(Cyto C) in mitochondria and the mRNA and protein expressions of GLS1 and proliferating nuclear antigen in cells(P<0.01). The mRNA expression of Bcl-2 and Bcl-xl, the protein expression of pro-caspase-9 and pro-caspase-3, and the ratio of Bcl-2/Bax and Bcl-xl/Bad in cells were decreased(P<0.01). Cyto C concentration in the cytoplasm, the mRNA expressions of Bax, Bad, apoptosis protease activating factor 1(Apaf-1), and protein expressions of cleaved-caspase-9, cleaved-caspase-3, cleaved-PARP-1 were increased(P<0.01). The aforementioned results indicate that TCS can counteract the senescent GES-1 cells induced by D-gal, and its mechanism may be closely related to suppressing the Gln/GLS1/α-KG metabolic axis, activating the mitochondrial apoptosis pathway, and thereby accelerating the apoptosis of the senescent cells and eliminating senescent cells.
Subject(s)
Apoptosis , Fruit , Galactose , Glutaminase , Glutamine , Mitochondria , Signal Transduction , Triterpenes , Apoptosis/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Triterpenes/pharmacology , Triterpenes/chemistry , Humans , Signal Transduction/drug effects , Cell Line , Fruit/chemistry , Glutamine/pharmacology , Glutamine/metabolism , Glutaminase/metabolism , Glutaminase/genetics , Cellular Senescence/drug effects , Ketoglutaric Acids/pharmacology , Ketoglutaric Acids/metabolismABSTRACT
Nonheme Fe(II) and 2-oxoglutarate (2OG)-dependent histone lysine demethylases 2A (KDM2A) catalyze the demethylation of the mono- or dimethylated lysine 36 residue in the histone H3 peptide (H3K36me1/me2), which plays a crucial role in epigenetic regulation and can be involved in many cancers. Although the overall catalytic mechanism of KDMs has been studied, how KDM2 catalysis takes place in contrast to other KDMs remains unknown. Understanding such differences is vital for enzyme redesign and can help in enzyme-selective drug design. Herein, we employed molecular dynamics (MD) and combined quantum mechanics/molecular mechanics (QM/MM) to explore the complete catalytic mechanism of KDM2A, including dioxygen diffusion and binding, dioxygen activation, and substrate oxidation. Our study demonstrates that the catalysis of KDM2A is controlled by the conformational change of the second coordination sphere (SCS), specifically by a change in the orientation of Y222, which unlocks the 2OG rearrangement from off-line to in-line mode. The study demonstrates that the variant Y222A makes the 2OG rearrangement more favorable. Furthermore, the study reveals that it is the size of H3K36me3 that prevents the 2OG rearrangement, thus rendering the enzyme inactivity with trimethylated lysine. Calculations show that the SCS and long-range interacting residues that stabilize the HAT transition state in KDM2A differ from those in KDM4A, KDM7B, and KDM6A, thus providing the basics for the enzyme-selective redesign and modulation of KDM2A without influencing other KDMs.
Subject(s)
Jumonji Domain-Containing Histone Demethylases , Molecular Dynamics Simulation , Jumonji Domain-Containing Histone Demethylases/metabolism , Jumonji Domain-Containing Histone Demethylases/chemistry , Humans , Ketoglutaric Acids/chemistry , Ketoglutaric Acids/metabolism , Oxygen/chemistry , Oxygen/metabolism , Biocatalysis , Quantum Theory , Ferrous Compounds/chemistry , Ferrous Compounds/metabolism , F-Box ProteinsABSTRACT
PURPOSE: To identify therapies for combined D, L-2-hydroxyglutaric aciduria (C-2HGA), a rare genetic disorder caused by recessive variants in the SLC25A1 gene. METHODS: Patients C-2HGA were identified and diagnosed by whole exome sequencing and biochemical genetic testing. Patient derived fibroblasts were then treated with phenylbutyrate and the functional effects assessed by metabolomics and RNA-sequencing. RESULTS: In this study, we demonstrated that C-2HGA patient derived fibroblasts exhibited impaired cellular bioenergetics. Moreover, Fibroblasts form one patient exhibited worsened cellular bioenergetics when supplemented with citrate. We hypothesized that treating patient cells with phenylbutyrate (PB), an FDA approved pharmaceutical drug that conjugates glutamine for renal excretion, would reduce mitochondrial 2-ketoglutarate, thereby leading to improved cellular bioenergetics. Metabolomic and RNA-seq analyses of PB-treated fibroblasts demonstrated a significant decrease in intracellular 2-ketoglutarate, 2-hydroxyglutarate, and in levels of mRNA coding for citrate synthase and isocitrate dehydrogenase. Consistent with the known action of PB, an increased level of phenylacetylglutamine in patient cells was consistent with the drug acting as 2-ketoglutarate sink. CONCLUSION: Our pre-clinical studies suggest that citrate supplementation has the possibility exacerbating energy metabolism in this condition. However, improvement in cellular bioenergetics suggests phenylbutyrate might have interventional utility for this rare disease.
Subject(s)
Fibroblasts , Glutarates , Phenylbutyrates , Humans , Phenylbutyrates/pharmacology , Phenylbutyrates/therapeutic use , Fibroblasts/metabolism , Fibroblasts/drug effects , Glutarates/metabolism , Ketoglutaric Acids/metabolism , Energy Metabolism/drug effects , Energy Metabolism/genetics , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondria/genetics , Metabolomics , Exome Sequencing , Citrate (si)-Synthase/metabolism , Citrate (si)-Synthase/genetics , Brain Diseases, Metabolic, Inborn/drug therapy , Brain Diseases, Metabolic, Inborn/genetics , Brain Diseases, Metabolic, Inborn/metabolism , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/metabolism , Brain Diseases, Metabolic/drug therapy , Brain Diseases, Metabolic/genetics , Brain Diseases, Metabolic/metabolism , Brain Diseases, Metabolic/pathology , Multiomics , Mitochondrial Proteins , Organic Anion TransportersABSTRACT
Potentiation of the effects of currently available antibiotics is urgently required to tackle the rising antibiotics resistance. The pyruvate (P) cycle has been shown to play a critical role in mediating aminoglycoside antibiotic killing, but the mechanism remains unexplored. In this study, we investigated the effects of intermediate metabolites of the P cycle regarding the potentiation of gentamicin. We found that α-ketoglutarate (α-KG) has the best synergy with gentamicin compared to the other metabolites. This synergistic killing effect was more effective with aminoglycosides than other types of antibiotics, and it was effective against various types of bacterial pathogens. Using fish and mouse infection models, we confirmed that the synergistic killing effect occurred in vivo. Furthermore, functional proteomics showed that α-KG downregulated thiosulphate metabolism. Upregulation of thiosulphate metabolism by exogenous thiosulphate counteracted the killing effect of gentamicin. The role of thiosulphate metabolism in antibiotic resistance was further confirmed using thiosulphate reductase knockout mutants. These mutants were more sensitive to gentamicin killing, and less tolerant to antibiotics compared to their parental strain. Thus, our study highlights a strategy for potentiating antibiotic killing by using a metabolite that reduces antibiotic resistance.
Subject(s)
Anti-Bacterial Agents , Gentamicins , Ketoglutaric Acids , Anti-Bacterial Agents/pharmacology , Animals , Ketoglutaric Acids/metabolism , Ketoglutaric Acids/pharmacology , Mice , Gentamicins/pharmacology , Drug Synergism , Microbial Sensitivity Tests , Bacteria/drug effects , Bacteria/genetics , Bacteria/metabolism , Down-Regulation/drug effects , Bacterial Infections/drug therapy , Bacterial Infections/microbiology , Disease Models, AnimalABSTRACT
The hydroxylation of remote C(sp3)-H bonds in aliphatic amino acids yields crucial precursors for the synthesis of high-value compounds. However, accurate regulation of the regioselectivity of remote C(sp3)-H bonds hydroxylation in aliphatic amino acids continues to be a common challenge in chemosynthesis and biosynthesis. In this study, the Fe(II)/α-ketoglutarate-dependent dioxygenase from Bacillus subtilis (BlAH) was mined and found to catalyze hydroxylation at the γ and δ sites of aliphatic amino acids. Crystal structure analysis, molecular dynamics simulations, and quantum chemical calculations revealed that regioselectivity was regulated by the spatial effect of BlAH. Based on these results, the spatial effect of BlAH was reconstructed to stabilize the transition state at the δ site of aliphatic amino acids, thereby successfully reversing the γ site regioselectivity to the δ site. For example, the regioselectivity of L-Homoleucine (5 a) was reversed from the γ site (1 : 12) to the δ site (>99 : 1). The present study not only expands the toolbox of biocatalysts for the regioselective functionalization of remote C(sp3)-H bonds, but also provides a theoretical guidance for the precision-driven modification of similarly remote C(sp3)-H bonds in complex molecules.
Subject(s)
Amino Acids , Bacillus subtilis , Dioxygenases , Ketoglutaric Acids , Hydroxylation , Bacillus subtilis/enzymology , Dioxygenases/metabolism , Dioxygenases/chemistry , Ketoglutaric Acids/metabolism , Ketoglutaric Acids/chemistry , Amino Acids/chemistry , Amino Acids/metabolism , Stereoisomerism , Ferrous Compounds/chemistry , Ferrous Compounds/metabolism , Molecular Dynamics SimulationABSTRACT
Studies on antiaging remedies in insect models sometimes show discrepancies in results. These discrepancies could be explained by different responses of short- and long-lived strains on the antiaging remedies. The purpose of the study was to test whether life-prolonging effects of alpha-ketoglutarate (AKG), observed in nematodes and fruit flies, would be reproduced in long-lived Drosophila melanogaster flies. Lifespan was assayed in flies kept in demographic cages. Fecundity, proportion of flies capable of negative geotaxis, starvation resistance, time of heat coma onset, levels of triacyglycerols, body glucose, glycogen, activities of glutamate dehydrogenase, catalase, glutathione-S-transferase, hexokinase, phosphofructokinase, pyruvate kinase, lactate, and glutamate dehydrogenases were assessed. Dietary AKG did not affect fly lifespan on the diet with 5% yeast and 5% sucrose (5Y:5S) and on the diet with 9% yeast and 1% sucrose (9Y:1S), but increased lifespan on the low-protein diet (1Y:9S). Twenty-five-day-old female flies fed a 5Y:5S diet with 10 mM AKG for 3 weeks, did not differ from the control group (without AKG) in climbing activity, resistance to heat stress, and starvation. The levels of glucose and glycogen were unaffected but the levels of triacylglycerols were lower in AKG-fed female flies. No differences in activities of glycolytic enzymes, NADPH-producing enzymes, glutamate dehydrogenase, oxygen consumption, and levels of oxidative stress markers were observed between the control and AKG-fed flies. However, AKG-fed flies had lower activities of catalase and glutathione-S-transferase. These results suggest that potential antiaging remedies, such as AKG, may not extend lifespan in long-living organisms despite influencing several metabolic parameters.
Subject(s)
Drosophila melanogaster , Ketoglutaric Acids , Longevity , Animals , Drosophila melanogaster/physiology , Drosophila melanogaster/drug effects , Drosophila melanogaster/metabolism , Longevity/drug effects , Ketoglutaric Acids/pharmacology , Ketoglutaric Acids/metabolism , Female , Male , Dietary SupplementsABSTRACT
The application of adipose-derived stem cells (ADSCs) in treating hard-to-heal wounds has been widely accepted, while the short-term survival rate remains an obstacle in stem cell therapy. The aim of this study is to investigate the effect of preconditioning ADSCs with α-ketoglutarate (α-KG) on the healing of acid burn wounds and cell survival within wounds. Preconditioning of ADSCs was performed by treating cells at passage 3 with 3.5 mM DM-αKG for 24 h. Proliferation and migration of ADSCs was examined. An acid burn wound was created on the dorsal skin of mice. Cell suspension of ADSCs (2 × 106 cells/ml), either pre-treated with α-KG or not, was injected subcutaneously around the margin of wound. At 1,4,7,10,14 days after injection, the percentage of wound closure was evaluated. Expression of pro-angiogenic factors, matrix molecules and HIF1-α in pretreated ADSCs or in wounds was evaluated by qRT-PCR and immunohistochemistry staining, respectively. The survival rate of DiO-labelled ADSCs was determined with the in vivo bioluminescent imaging system. Treating with α-KG induced an enhancement in migration of ADSCs, while their proliferation was not affected. Expression of Vegf and Fgf-2 was significantly increased. With injection of pretreated ADSCs, healing of wounds was remarkably accelerated, along with increased ECM deposition and microvessel density. Moreover, pretreatment with α-KG resulted a prolonged survival of engrafted ADSCs was observed. Expression of HIF-1α was significantly increased in ADSCs treated with α-KG and in wounds injected with preconditioned ADSCs. Our results revealed that healing of acid burn wound was accelerated with administration of ADSCs pretreated with α-KG, which induced elevated expression of HIF-1α and prolonged survival of engrafted stem cells.
Subject(s)
Adipose Tissue , Burns , Ketoglutaric Acids , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Wound Healing , Animals , Wound Healing/drug effects , Ketoglutaric Acids/metabolism , Ketoglutaric Acids/pharmacology , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Burns/therapy , Burns/pathology , Mice , Adipose Tissue/cytology , Mesenchymal Stem Cell Transplantation/methods , Cell Survival/drug effects , Cell Proliferation/drug effects , Male , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Cell Movement/drug effects , Cells, CulturedABSTRACT
Hydroxyectoine is an important compatible solute that holds potential for development into a high-value chemical with broad applications. However, the traditional high-salt fermentation for hydroxyectoine production presents challenges in treating the high-salt wastewater. Here, we report the rational engineering of Halomonas salifodinae to improve the bioproduction of hydroxyectoine under lower-salt conditions. The comparative transcriptomic analysis suggested that the increased expression of ectD gene encoding ectoine hydroxylase (EctD) and the decreased expressions of genes responsible for tricarboxylic acid (TCA) cycle contributed to the increased hydroxyectoine production in H. salifodinae IM328 grown under high-salt conditions. By blocking the degradation pathway of ectoine and hydroxyectoine, enhancing the expression of ectD, and increasing the supply of 2-oxoglutarate, the engineered H. salifodinae strain HS328-YNP15 (ΔdoeA::PUP119-ectD p-gdh) produced 8.3-fold higher hydroxyectoine production than the wild-type strain and finally achieved a hydroxyectoine titer of 4.9 g/L in fed-batch fermentation without any detailed process optimization. This study shows the potential to integrate hydroxyectoine production into open unsterile fermentation process that operates under low-salinity and high-alkalinity conditions, paving the way for next-generation industrial biotechnology. KEY POINTS: ⢠Hydroxyectoine production in H. salifodinae correlates with the salinity of medium ⢠Transcriptomic analysis reveals the limiting factors for hydroxyectoine production ⢠The engineered strain produced 8.3-fold more hydroxyectoine than the wild type.
Subject(s)
Amino Acids, Diamino , Fermentation , Halomonas , Metabolic Engineering , Halomonas/genetics , Halomonas/metabolism , Metabolic Engineering/methods , Amino Acids, Diamino/biosynthesis , Amino Acids, Diamino/metabolism , Amino Acids, Diamino/genetics , Citric Acid Cycle/genetics , Gene Expression Profiling , Sodium Chloride/metabolism , Salinity , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Ketoglutaric Acids/metabolismABSTRACT
Proline-5-carboxylate reductase 2, encoded by PYCR2 gene, is an enzyme that catalyzes the last step of proline synthesis from pyrroline-5-carboxylate synthetase to proline. PYCR2 gene defect causes hypomyelinating leukodystrophy 10. Up until now, to our knowledge around 38 patients with PYCR2 defect have been reported. Herein, we describe clinical, neuroradiological, biochemical findings, and metabolomic profiling of three new genetically related cases of PYCR2 defects from a large family. Cerebrospinal fluid (CSF) amino acid levels were measured and untargeted metabolomic profiling of plasma and CSF were conducted and evaluated together with the clinical findings in the patients. While plasma and CSF proline levels were found to be totally normal, untargeted metabolomic profiling revealed mild increases of glutamate, alpha-ketoglutarate, and l-glutamate semialdehyde and marked increases of inosine and xanthine. Our findings and all the previous reports suggest that proline auxotrophy is not the central disease mechanism. Untargeted metabolomics point to mild changes in proline pathway and also in purine/pyrimidine pathway.