RESUMEN
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.
Asunto(s)
Metabolismo Energético , Células Epiteliales , Helicobacter pylori , Histonas , Neoplasias Gástricas , gamma-Glutamiltransferasa , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/genética , Animales , Histonas/metabolismo , Metilación , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/patología , gamma-Glutamiltransferasa/metabolismo , gamma-Glutamiltransferasa/genética , Ratones , Humanos , Ratones Desnudos , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Proliferación Celular , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Infecciones por Helicobacter/complicaciones , Ácidos Cetoglutáricos/metabolismoRESUMEN
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.
Asunto(s)
Ácidos Cetoglutáricos , Síndrome Metabólico , Síndrome Metabólico/metabolismo , Ácidos Cetoglutáricos/metabolismo , Humanos , Obesidad/metabolismo , Obesidad/complicaciones , Animales , Estrés OxidativoRESUMEN
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.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Azoximetano , Carcinogénesis , Neoplasias Colorrectales , Metilación de ADN , Dioxigenasas , Animales , Humanos , Ratones , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Azoximetano/toxicidad , Azoximetano/efectos adversos , Células CACO-2 , Carcinogénesis/genética , Colitis/inducido químicamente , Colitis/genética , Colitis/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/inducido químicamente , Neoplasias Colorrectales/etiología , Sulfato de Dextran/toxicidad , Dioxigenasas/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Ácidos Cetoglutáricos/metabolismo , Ratones Noqueados , Homólogo 1 de la Proteína MutL/genética , Homólogo 1 de la Proteína MutL/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
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.
Asunto(s)
Ferroptosis , Ácidos Cetoglutáricos , Osteoartritis , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Transducción de Señal , Ferroptosis/efectos de los fármacos , Animales , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismo , Osteoartritis/patología , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/farmacología , Transducción de Señal/efectos de los fármacos , Ratas , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Sistema de Transporte de Aminoácidos y+/metabolismo , Sistema de Transporte de Aminoácidos y+/genética , Masculino , Proteínas Proto-Oncogénicas c-ets/metabolismo , Proteínas Proto-Oncogénicas c-ets/genética , Ratas Sprague-Dawley , Apoptosis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
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.
Asunto(s)
Hierro , Ácidos Cetoglutáricos , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/química , Hierro/metabolismo , Hierro/química , Hidroxilación , Ciclización , Oxigenasas/metabolismo , Oxigenasas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/químicaRESUMEN
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.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Hiperlipidemias , Ácidos Cetoglutáricos , Factor 2 Relacionado con NF-E2 , Estrés Oxidativo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Transducción de Señal , Animales , Hiperlipidemias/metabolismo , Hiperlipidemias/tratamiento farmacológico , Hiperlipidemias/complicaciones , Ratones , Estrés Oxidativo/efectos de los fármacos , Humanos , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/farmacología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Transducción de Señal/efectos de los fármacos , Células Hep G2 , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Masculino , Metabolismo de los Lípidos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Hígado Graso/metabolismo , Hígado Graso/etiología , Hígado Graso/tratamiento farmacológico , Hígado Graso/prevención & control , Hígado Graso/patología , Modelos Animales de Enfermedad , Hígado/metabolismo , Hígado/efectos de los fármacos , Hígado/patologíaRESUMEN
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.
Asunto(s)
Bacillus subtilis , Carbono , Ciclo del Ácido Cítrico , Isocitrato Deshidrogenasa , Ácidos Cetoglutáricos , Malato Deshidrogenasa , Nitrógeno , Nitrógeno/metabolismo , Carbono/metabolismo , Malato Deshidrogenasa/metabolismo , Malato Deshidrogenasa/genética , Bacillus subtilis/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/enzimología , Isocitrato Deshidrogenasa/metabolismo , Isocitrato Deshidrogenasa/genética , Ácidos Cetoglutáricos/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Compuestos de Amonio/metabolismoRESUMEN
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.
Asunto(s)
Colágeno , Matriz Extracelular , Ácido Láctico , Metástasis de la Neoplasia , Neoplasias de la Próstata , Masculino , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/genética , Humanos , Matriz Extracelular/metabolismo , Colágeno/metabolismo , Ácido Láctico/metabolismo , Línea Celular Tumoral , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Transducción de Señal , Regulación Neoplásica de la Expresión Génica , Animales , Ratones , Microambiente Tumoral , Ácidos Cetoglutáricos/metabolismo , HidroxilaciónRESUMEN
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.
Asunto(s)
Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Hierro , Ácidos Cetoglutáricos , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/química , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/química , Hierro/metabolismo , Hierro/química , Humanos , Especificidad por Sustrato , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/química , Conformación Proteica , Uracilo/metabolismo , Uracilo/análogos & derivados , Uracilo/química , Simulación de Dinámica Molecular , Timina/análogos & derivadosRESUMEN
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.
Asunto(s)
Apoptosis , Frutas , Galactosa , Glutaminasa , Glutamina , Mitocondrias , Transducción de Señal , Triterpenos , Apoptosis/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Triterpenos/farmacología , Triterpenos/química , Humanos , Transducción de Señal/efectos de los fármacos , Línea Celular , Frutas/química , Glutamina/farmacología , Glutamina/metabolismo , Glutaminasa/metabolismo , Glutaminasa/genética , Senescencia Celular/efectos de los fármacos , Ácidos Cetoglutáricos/farmacología , Ácidos Cetoglutáricos/metabolismoRESUMEN
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.
Asunto(s)
Histona Demetilasas con Dominio de Jumonji , Simulación de Dinámica Molecular , Histona Demetilasas con Dominio de Jumonji/metabolismo , Histona Demetilasas con Dominio de Jumonji/química , Humanos , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Oxígeno/química , Oxígeno/metabolismo , Biocatálisis , Teoría Cuántica , Compuestos Ferrosos/química , Compuestos Ferrosos/metabolismo , Proteínas F-BoxRESUMEN
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.
Asunto(s)
Fibroblastos , Glutaratos , Fenilbutiratos , Humanos , Fenilbutiratos/farmacología , Fenilbutiratos/uso terapéutico , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Glutaratos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/genética , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/genética , Metabolómica , Secuenciación del Exoma , Citrato (si)-Sintasa/metabolismo , Citrato (si)-Sintasa/genética , Encefalopatías Metabólicas Innatas/tratamiento farmacológico , Encefalopatías Metabólicas Innatas/genética , Encefalopatías Metabólicas Innatas/metabolismo , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Encefalopatías Metabólicas/tratamiento farmacológico , Encefalopatías Metabólicas/genética , Encefalopatías Metabólicas/metabolismo , Encefalopatías Metabólicas/patología , Multiómica , Proteínas Mitocondriales , Transportadores de Anión OrgánicoRESUMEN
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.
Asunto(s)
Antibacterianos , Gentamicinas , Ácidos Cetoglutáricos , Antibacterianos/farmacología , Animales , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/farmacología , Ratones , Gentamicinas/farmacología , Sinergismo Farmacológico , Pruebas de Sensibilidad Microbiana , Bacterias/efectos de los fármacos , Bacterias/genética , Bacterias/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/microbiología , Modelos Animales de EnfermedadRESUMEN
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.
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Aminoácidos , Bacillus subtilis , Dioxigenasas , Ácidos Cetoglutáricos , Hidroxilación , Bacillus subtilis/enzimología , Dioxigenasas/metabolismo , Dioxigenasas/química , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/química , Aminoácidos/química , Aminoácidos/metabolismo , Estereoisomerismo , Compuestos Ferrosos/química , Compuestos Ferrosos/metabolismo , Simulación de Dinámica MolecularRESUMEN
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.
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Drosophila melanogaster , Ácidos Cetoglutáricos , Longevidad , Animales , Drosophila melanogaster/fisiología , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/metabolismo , Longevidad/efectos de los fármacos , Ácidos Cetoglutáricos/farmacología , Ácidos Cetoglutáricos/metabolismo , Femenino , Masculino , Suplementos DietéticosRESUMEN
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.
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Tejido Adiposo , Quemaduras , Ácidos Cetoglutáricos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Cicatrización de Heridas , Animales , Cicatrización de Heridas/efectos de los fármacos , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/farmacología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Quemaduras/terapia , Quemaduras/patología , Ratones , Tejido Adiposo/citología , Trasplante de Células Madre Mesenquimatosas/métodos , Supervivencia Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Masculino , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Movimiento Celular/efectos de los fármacos , Células CultivadasRESUMEN
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.
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Aminoácidos Diaminos , Fermentación , Halomonas , Ingeniería Metabólica , Halomonas/genética , Halomonas/metabolismo , Ingeniería Metabólica/métodos , Aminoácidos Diaminos/biosíntesis , Aminoácidos Diaminos/metabolismo , Aminoácidos Diaminos/genética , Ciclo del Ácido Cítrico/genética , Perfilación de la Expresión Génica , Cloruro de Sodio/metabolismo , Salinidad , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Ácidos Cetoglutáricos/metabolismoRESUMEN
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.
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Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias , Metabolómica , Prolina , Pirrolina Carboxilato Reductasas , Niño , Femenino , Humanos , Masculino , delta-1-Pirrolina-5-Carboxilato Reductasa , Ácido Glutámico/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/patología , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/diagnóstico , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/sangre , Imagen por Resonancia Magnética , Redes y Vías Metabólicas/genética , Metaboloma/genética , Metabolómica/métodos , Mutación/genética , Linaje , Prolina/líquido cefalorraquídeo , Purinas/metabolismo , Pirimidinas , Pirrolina Carboxilato Reductasas/genética , Pirrolina Carboxilato Reductasas/deficiencia , Xantina/sangre , LactanteRESUMEN
AIMS: Metabolic reprogramming in cancer cells has been linked to mitochondrial dysfunction. The mitochondrial 2-oxoglutarate/malate carrier (OGC) has been suggested as a potential target for preventing cancer progression. Although OGC is involved in the malate/aspartate shuttle, its exact role in cancer metabolism remains unclear. We aimed to investigate whether OGC may contribute to the alteration of mitochondrial inner membrane potential by transporting protons. METHODS: The expression of OGC in mouse tissues and cancer cells was investigated by PCR and Western blot analysis. The proton transport function of recombinant murine OGC was evaluated by measuring the membrane conductance (Gm) of planar lipid bilayers. OGC-mediated substrate transport was measured in proteoliposomes using 14C-malate. RESULTS: OGC increases proton Gm only in the presence of natural (long-chain fatty acids, FA) or chemical (2,4-dinitrophenol) protonophores. The increase in OGC activity directly correlates with the increase in the number of unsaturated bonds of the FA. OGC substrates and inhibitors compete with FA for the same protein binding site. Arginine 90 was identified as a critical amino acid for the binding of FA, ATP, 2-oxoglutarate, and malate, which is a first step towards understanding the OGC-mediated proton transport mechanism. CONCLUSION: OGC extends the family of mitochondrial transporters with dual function: (i) metabolite transport and (ii) proton transport facilitated in the presence of protonophores. Elucidating the contribution of OGC to uncoupling may be essential for the design of targeted drugs for the treatment of cancer and other metabolic diseases.
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2,4-Dinitrofenol , Ácidos Grasos , Animales , 2,4-Dinitrofenol/farmacología , Ratones , Ácidos Grasos/metabolismo , Humanos , Malatos/metabolismo , Mitocondrias/metabolismo , Transporte Iónico/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Protones , Ácidos Cetoglutáricos/metabolismo , Transportadores de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico/genética , Proteínas de Transporte de MembranaRESUMEN
In plants, salicylic acid (SA) hydroxylation regulates SA homoeostasis, playing an essential role during plant development and response to pathogens. This reaction is catalysed by SA hydroxylase enzymes, which hydroxylate SA producing 2,3-dihydroxybenzoic acid (2,3-DHBA) and/or 2,5-dihydroxybenzoic acid (2,5-DHBA). Several SA hydroxylases have recently been identified and characterised from different plant species, but no such activity has yet been reported in maize. In this work, we describe the identification and characterisation of a new SA hydroxylase in maize plants. This enzyme, with high sequence similarity to previously described SA hydroxylases from Arabidopsis and rice, converts SA into 2,5-DHBA; however, it has different kinetic properties to those of previously characterised enzymes, and it also catalysers the conversion of the flavonoid dihydroquercetin into quercetin in in vitro activity assays, suggesting that the maize enzyme may have different roles in vivo to those previously reported from other species. Despite this, ZmS5H can complement the pathogen resistance and the early senescence phenotypes of Arabidopsis s3h mutant plants. Finally, we characterised a maize mutant in the S5H gene (s5hMu) that has altered growth, senescence and increased resistance against Colletotrichum graminicola infection, showing not only alterations in SA and 2,5-DHBA but also in flavonol levels. Together, the results presented here provide evidence that SA hydroxylases in different plant species have evolved to show differences in catalytic properties that may be important to fine tune SA levels and other phenolic compounds such as flavonols, to regulate different aspects of plant development and pathogen defence.