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1.
J Am Chem Soc ; 146(22): 15155-15166, 2024 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-38775806

RESUMEN

Fructose-1,6-bisphosphate (FBP), a cellular endogenous sugar metabolite in the glycolytic pathway, has recently been reported to act as a signaling molecule to regulate various cellular events through the engagement of important proteins. Though tremendous progress has been made in identifying specific FBP-protein interactions, the comprehensive identification of FBP-interacting proteins and their regulatory mechanisms remains largely unexplored. Here, we describe a concise synthetic approach for the scalable preparation of a photoaffinity FBP probe that enables the quantitative chemoproteomic profiling of FBP-protein interactions based on photoaffinity labeling (PAL) directly in living cells. Using such a protocol, we captured known FBP targets including PKM2 and MDH2. Furthermore, among unknown FBP-interacting proteins, we identified a mitochondrial metabolic enzyme aldehyde dehydrogenase 2 (ALDH2), against which FBP showed inhibitory activity and resulted in cellular ROS upregulation accompanied by mitochondrial fragmentation. Our findings disclosed a new mode of glucose signaling mediating by the FBP-ALDH2-ROS axis.


Asunto(s)
Aldehído Deshidrogenasa Mitocondrial , Fructosadifosfatos , Proteómica , Humanos , Fructosadifosfatos/metabolismo , Aldehído Deshidrogenasa Mitocondrial/metabolismo , Transducción de Señal , Especies Reactivas de Oxígeno/metabolismo , Mitocondrias/metabolismo
2.
BMC Microbiol ; 24(1): 151, 2024 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-38702601

RESUMEN

BACKGROUND: Fluoride-resistant Streptococcus mutans (S. mutans) strains have developed due to the wide use of fluoride in dental caries prevention. However, the metabolomics of fluoride-resistant S. mutans remains unclear. OBJECTIVE: This study aimed to identify metabolites that discriminate fluoride-resistant from wild-type S. mutans. MATERIALS AND METHODS: Cell supernatants from fluoride-resistant and wild-type S. mutans were collected and analyzed by liquid chromatography-mass spectrometry. Principal components analysis and partial least-squares discriminant analysis were performed for the statistical analysis by variable influence on projection (VIP > 2.0) and p value (Mann-Whitney test, p < 0.05). Metabolites were assessed qualitatively using the Human Metabolome Database version 2.0 ( http://www.hmdb.ca ), or Kyoto Encyclopedia of Genes and Genomes ( http://www.kegg.jp ), and Metaboanalyst 6.0 ( https://www.metaboanalyst.ca ). RESULTS: Fourteen metabolites differed significantly between fluoride-resistant and wild-type strains in the early log phase. Among these metabolites, 5 were identified. There were 32 differential metabolites between the two strains in the stationary phase, 13 of which were identified. The pyrimidine metabolism for S. mutans FR was matched with the metabolic pathway. CONCLUSIONS: The fructose-1,6-bisphosphate concentration increased in fluoride-resistant strains under acidic conditions, suggesting enhanced acidogenicity and acid tolerance. This metabolite may be a promising target for elucidating the cariogenic and fluoride resistant mechanisms of S. mutans.


Asunto(s)
Farmacorresistencia Bacteriana , Fluoruros , Fructosadifosfatos , Metabolómica , Streptococcus mutans , Streptococcus mutans/efectos de los fármacos , Streptococcus mutans/genética , Streptococcus mutans/metabolismo , Metabolómica/métodos , Fluoruros/metabolismo , Fluoruros/farmacología , Fructosadifosfatos/metabolismo , Humanos , Metaboloma/efectos de los fármacos , Caries Dental/microbiología , Cromatografía Liquida
3.
Adv Sci (Weinh) ; 10(7): e2203528, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36642839

RESUMEN

Metabolites are important for cell fate determination. Fructose-1,6-bisphosphate (F1,6P) is the rate-limiting product in glycolysis and the rate-limiting substrate in gluconeogenesis. Here, it is discovered that the nuclear-accumulated F1,6P impairs cancer cell viability by directly binding to high mobility group box 1 (HMGB1), the most abundant non-histone chromosome structural protein with paradoxical roles in tumor development. F1,6P disrupts the association between the HMGB1 A-box and C-tail by targeting K43/K44 residues, inhibits HMGB1 oligomerization, and stabilizes P53 protein by increasing P53-HMGB1 interaction. Moreover, F1,6P lowers the affinity of HMGB1 for DNA and DNA adducts, which sensitizes cancer cells to chemotherapeutic drug(s)-induced DNA replication stress and DNA damage. Concordantly, F1,6P resensitizes cancer cells with chemotherapy resistance, impairs tumor growth and enhances chemosensitivity in mice, and impedes the growth of human tumor organoids. These findings reveal a novel role for nuclear-accumulated F1,6P and underscore the potential utility of F1,6P as a drug for cancer therapy.


Asunto(s)
Fructosadifosfatos , Proteína HMGB1 , Neoplasias , Animales , Humanos , Ratones , Daño del ADN , Glucólisis , Proteína HMGB1/química , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Neoplasias/metabolismo , Proteína p53 Supresora de Tumor/genética , Fructosadifosfatos/metabolismo
4.
Proc Natl Acad Sci U S A ; 119(31): e2204407119, 2022 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-35881794

RESUMEN

Cellular metabolism is regulated over space and time to ensure that energy production is efficiently matched with consumption. Fluorescent biosensors are useful tools for studying metabolism as they enable real-time detection of metabolite abundance with single-cell resolution. For monitoring glycolysis, the intermediate fructose 1,6-bisphosphate (FBP) is a particularly informative signal as its concentration is strongly correlated with flux through the whole pathway. Using GFP insertion into the ligand-binding domain of the Bacillus subtilis transcriptional regulator CggR, we developed a fluorescent biosensor for FBP termed HYlight. We demonstrate that HYlight can reliably report the real-time dynamics of glycolysis in living cells and tissues, driven by various metabolic or pharmacological perturbations, alone or in combination with other physiologically relevant signals. Using this sensor, we uncovered previously unknown aspects of ß-cell glycolytic heterogeneity and dynamics.


Asunto(s)
Técnicas Biosensibles , Fructosa , Glucólisis , Análisis de la Célula Individual , Fluorescencia , Fructosa/análisis , Fructosadifosfatos/análisis , Humanos , Células Secretoras de Insulina/química , Células Secretoras de Insulina/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Análisis de la Célula Individual/métodos
5.
Aging (Albany NY) ; 14(7): 3233-3258, 2022 04 11.
Artículo en Inglés | MEDLINE | ID: mdl-35404841

RESUMEN

Metabolic reprogramming and elevated glycolysis levels are associated with tumor progression. However, despite cancer cells selectively inhibiting or expressing certain metabolic enzymes, it is unclear whether differences in gene profiles influence patient outcomes. Therefore, identifying the differences in enzyme action may facilitate discovery of gene ontology variations to characterize tumors. Fructose-1,6-bisphosphate (F-1,6-BP) is an important intermediate in glucose metabolism, particularly in cancer. Gluconeogenesis and glycolysis require fructose-1,6-bisphosphonates 1 (FBP1) and fructose-bisphosphate aldolase A (ALDOA), which participate in F-1,6-BP conversion. Increased expression of ALDOA and decreased expression of FBP1 are associated with the progression of various forms of cancer in humans. However, the exact molecular mechanism by which ALDOA and FBP1 are involved in the switching of F-1,6-BP is not yet known. As a result of their pancancer pattern, the relationship between ALDOA and FBP1 in patient prognosis is reversed, particularly in lung adenocarcinoma (LUAD) and liver hepatocellular carcinoma (LIHC). Using The Cancer Genome Atlas (TCGA), we observed that FBP1 expression was low in patients with LUAD and LIHC tumors, which was distinct from ALDOA. A similar trend was observed in the analysis of Cancer Cell Line Encyclopedia (CCLE) datasets. By dissecting downstream networks and possible upstream regulators, using ALDOA and FBP1 as the core, we identified common signatures and interaction events regulated by ALDOA and FBP1. Notably, the identified effectors dominated by ALDOA or FBP1 were distributed in opposite patterns and can be considered independent prognostic indicators for patients with LUAD and LIHC. Therefore, uncovering the effectors between ALDOA and FBP1 will lead to novel therapeutic strategies for cancer patients.


Asunto(s)
Adenocarcinoma del Pulmón , Carcinoma Hepatocelular , Fructosa-Bifosfato Aldolasa , Neoplasias Pulmonares , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Fructosa , Fructosa-Bifosfatasa/genética , Fructosa-Bifosfato Aldolasa/genética , Fructosa-Bifosfato Aldolasa/metabolismo , Fructosadifosfatos , Gluconeogénesis/genética , Glucólisis/genética , Humanos , Neoplasias Pulmonares/genética , Pronóstico
6.
Sci Rep ; 12(1): 304, 2022 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-34997135

RESUMEN

To evaluate the effects of fructose diphosphate (FDP) on routine coagulation tests in vitro, we added FDP into the mixed normal plasma to obtain the final concentration of 0, 1, 2, 3, 4, 5, 6, 10, 15, 20, 25, 30 and 35 mg/mL of drug. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen (FBG) and thrombin time (TT) of samples were analyzed with blood coagulation analyzers from four different manufacturers(Sysmex, Stago, SEKISUI and Werfen) and their corresponding reagents, respectively. Before the experiment, we also observed whether there were significant differences in coagulation test results of different lots of reagents produced by each manufacturer. At the same time as the four routine clotting tests, the Sysmex blood coagulation analyzer and its proprietary analysis software were used to detect the change of maximum platelet aggregation rate in platelet-rich plasma after adding FDP (0, 1, 2, 3, 4, 5 and 6 mg/mL). The results of PT, aPTT and TT showed a FDP (0-35 mg/mL) concentration-dependent increase and a FBG concentration-dependent decrease. The degree of change (increase or decrease) varied depending on the assay system, with PT and aPTT being more affected by the Sysmex blood coagulation testing instrument reagent system and less affected by CEKISUI, TT less affected by CEKISUI and more affected by Stago, and FBG less affected by Stago and more affected by Sysmex. The results of PT, aPTT and TT were statistically positively correlated with their FDP concentrations, while FBG was negatively correlated. The correlation coefficients between FDP and the coagulation testing systems of Sysmex, Stago, Werfen and SEKISUI were 0.975, 0.988, 0.967, 0.986 for PT, and 0.993, 0.989, 0.990 and 0.962 for aPTT, 0.994, 0.960, 0.977 and 0.982 for TT, - 0.990, - 0.983, - 0.989 and - 0.954 for FBG, respectively. Different concentrations of FDP (0, 1, 2, 3, 4, 5 and 6 mg/mL) had different effects on the maximum aggregation rate of platelet induced by the agonists of adenosine diphosphate (ADP, 5 µmol/L), arachidonic acid (Ara, 1 mmol/L), collagen (Col, 2.5 µg/mL) and epinephrine (Epi,10 µmol/L), but the overall downward trend was consistent, that is, with the increase of FDP concentration, the platelet aggregation rate decreased significantly. Our experimental study demonstrated a possible effect of FDP on the assays of coagulation and Platelet aggregation, which may arise because the drug interferes with the coagulation and platelet aggregation detection system, or it may affect our in vivo coagulation system and Platelet aggregation function, the real mechanism of which remains to be further verified and studied.


Asunto(s)
Pruebas de Coagulación Sanguínea , Coagulación Sanguínea/efectos de los fármacos , Fructosadifosfatos/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Tiempo de Tromboplastina Parcial , Agregación Plaquetaria/efectos de los fármacos , Pruebas de Función Plaquetaria , Tiempo de Protrombina , Tiempo de Trombina
7.
Cell Chem Biol ; 28(11): 1539-1541, 2021 11 18.
Artículo en Inglés | MEDLINE | ID: mdl-34798034

RESUMEN

Conversion of in vitro selected aptamers into functional metabolic sensors is hampered by reduced in vivo aptamer binding and limited tunability of cellular metabolite levels. In this issue of Cell Chemical Biology, Ortega et al. (2021) construct RNA sensors of fructose-6-bisphosphate (FBP) that report on metabolite levels within single yeast cells.


Asunto(s)
Fructosadifosfatos , Glucólisis , Colorantes , ARN , Sensación
8.
Nat Cell Biol ; 23(10): 1085-1094, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34616026

RESUMEN

Cells respond to stress by blocking translation, rewiring metabolism and forming transient messenger ribonucleoprotein assemblies called stress granules (SGs). After stress release, re-establishing homeostasis and disassembling SGs requires ATP-consuming processes. However, the molecular mechanisms whereby cells restore ATP production and disassemble SGs after stress remain poorly understood. Here we show that upon stress, the ATP-producing enzyme Cdc19 forms inactive amyloids, and that their rapid re-solubilization is essential to restore ATP production and disassemble SGs in glucose-containing media. Cdc19 re-solubilization is initiated by the glycolytic metabolite fructose-1,6-bisphosphate, which directly binds Cdc19 amyloids, allowing Hsp104 and Ssa2 chaperone recruitment and aggregate re-solubilization. Fructose-1,6-bisphosphate then promotes Cdc19 tetramerization, which boosts its activity to further enhance ATP production and SG disassembly. Together, these results describe a molecular mechanism that is critical for stress recovery and directly couples cellular metabolism with SG dynamics via the regulation of reversible Cdc19 amyloids.


Asunto(s)
Amiloide/química , Proteínas de Ciclo Celular/química , Gránulos Citoplasmáticos/química , Piruvato Quinasa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico , Adenosina Trifosfato/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fructosadifosfatos/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Piruvato Quinasa/química , Piruvato Quinasa/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
9.
Epilepsy Behav ; 122: 108223, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34388666

RESUMEN

Glucose metabolism is altered in epilepsy, and this may contribute to seizure generation. Recent research has shown that metabolic therapies including the ketogenic diet and medium chain triglycerides can improve energy metabolism in the brain. Fructose 1,6-bisphosphate (F16BP) is an intermediate of glycolysis and when administered exogenously is anticonvulsant in several rodent seizure models and may alter glucose metabolism. Here, we showed that F16BP elevated the seizure threshold in the acute 6-Hz mouse seizure model and investigated if F16BP could restore impairments in glucose metabolism occurring in the chronic stage of the pilocarpine mouse model of epilepsy. Two weeks after the pilocarpine injections, mice that experienced status epilepticus (SE, "epileptic") and did not experience SE (no SE, "nonepileptic") were injected with vehicle (0.9% saline) or F16BP (1 g/kg in 0.9% saline) daily for 5 consecutive days. At 3 weeks, mice were injected with [U-13C6]-glucose and the % enrichment of 13C in key metabolites in addition to the total levels of each metabolite was measured in the hippocampal formation and liver. Fructose 1,6-bisphosphate increased total GABA in the hippocampal formation, regardless of whether mice had experienced SE. In the hippocampal formation, F16BP prevented reductions in the % 13C enrichment of citrate, succinate, malate, glutamate, GABA and aspartate that occurred in the chronic stage of the pilocarpine model. Interestingly, % 13C enrichment in glucose-derived metabolites was reduced in the liver in the chronic stage of the pilocarpine model. Fructose 1,6-bisphosphate was also beneficial in the liver, preventing reductions in % 13C enrichment of lactate and alanine that were associated with SE. This study confirmed that F16BP is anticonvulsant and can improve elements of glucose metabolism that are dysregulated in the chronic stage of the pilocarpine model, which may be due to reduction of spontaneous seizures. Our results highlight that F16BP may be therapeutically beneficial for epilepsies refractory to treatment.


Asunto(s)
Epilepsia , Estado Epiléptico , Animales , Anticonvulsivantes/farmacología , Anticonvulsivantes/uso terapéutico , Modelos Animales de Enfermedad , Epilepsia/inducido químicamente , Epilepsia/tratamiento farmacológico , Fructosa/uso terapéutico , Fructosadifosfatos , Glucosa/metabolismo , Hipocampo , Hígado , Ratones , Estrés Oxidativo , Pilocarpina/toxicidad , Estado Epiléptico/tratamiento farmacológico
10.
Nat Commun ; 12(1): 4371, 2021 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-34272364

RESUMEN

Metabolic programming and mitochondrial dynamics along with T cell differentiation affect T cell fate and memory development; however, how to control metabolic reprogramming and mitochondrial dynamics in T cell memory development is unclear. Here, we provide evidence that the SUMO protease SENP1 promotes T cell memory development via Sirt3 deSUMOylation. SENP1-Sirt3 signalling augments the deacetylase activity of Sirt3, promoting both OXPHOS and mitochondrial fusion. Mechanistically, SENP1 activates Sirt3 deacetylase activity in T cell mitochondria, leading to reduction of the acetylation of mitochondrial metalloprotease YME1L1. Consequently, deacetylation of YME1L1 suppresses its activity on OPA1 cleavage to facilitate mitochondrial fusion, which results in T cell survival and promotes T cell memory development. We also show that the glycolytic intermediate fructose-1,6-bisphosphate (FBP) as a negative regulator suppresses AMPK-mediated activation of the SENP1-Sirt3 axis and reduces memory development. Moreover, glucose limitation reduces FBP production and activates AMPK during T cell memory development. These data show that glucose limitation activates AMPK and the subsequent SENP1-Sirt3 signalling for T cell memory development.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Linfocitos T CD8-positivos/inmunología , Cisteína Endopeptidasas/metabolismo , Memoria Inmunológica , Mitocondrias/metabolismo , Sirtuina 3/metabolismo , Linfocitos T/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Acetilación , Aloinjertos , Animales , Línea Celular Tumoral , Supervivencia Celular/genética , Neoplasias del Colon/inmunología , Fructosadifosfatos/metabolismo , GTP Fosfohidrolasas/metabolismo , Glucosa/deficiencia , Memoria Inmunológica/genética , Metabolómica , Metaloendopeptidasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Mitocondrias/genética , Mitocondrias/ultraestructura , Dinámicas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Fosforilación Oxidativa , Sirtuina 3/antagonistas & inhibidores , Sirtuina 3/genética , Sumoilación , Linfocitos T/inmunología
11.
Annu Rev Biochem ; 90: 31-55, 2021 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-34153217

RESUMEN

My graduate and postdoctoral training in metabolism and enzymology eventually led me to study the short- and long-term regulation of glucose and lipid metabolism. In the early phase of my career, my trainees and I identified, purified, and characterized a variety of phosphofructokinase enzymes from mammalian tissues. These studies led us to discover fructose 2,6-P2, the most potent activator of phosphofructokinase and glycolysis. The discovery of fructose 2,6-P2 led to the identification and characterization of the tissue-specific bifunctional enzyme 6-phosphofructo-2-kinase:fructose 2,6-bisphosphatase. We discovered a glucose signaling mechanism by which the liver maintains glucose homeostasis by regulating the activities of this bifunctional enzyme. With a rise in glucose, a signaling metabolite, xylulose 5-phosphate, triggers rapid activation of a specific protein phosphatase (PP2ABδC), which dephosphorylates the bifunctional enzyme, thereby increasing fructose 2,6-P2 levels and upregulating glycolysis. These endeavors paved the way for us to initiate the later phase of my career in which we discovered a new transcription factor termed the carbohydrate response element binding protein (ChREBP). Now ChREBP is recognized as the masterregulator controlling conversion of excess carbohydrates to storage of fat in the liver. ChREBP functions as a central metabolic coordinator that responds to nutrients independently of insulin. The ChREBP transcription factor facilitates metabolic adaptation to excess glucose, leading to obesity and its associated diseases.


Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Bioquímica/historia , Fructosadifosfatos/metabolismo , Fosfofructoquinasa-2/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/química , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Gluconeogénesis/fisiología , Glucosa/metabolismo , Glucólisis , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Masculino , Ratones , Fosfofructoquinasa-2/química , Fosfofructoquinasas/química , Fosfofructoquinasas/metabolismo , Fosforilación , Estados Unidos
12.
Trends Endocrinol Metab ; 32(8): 540-543, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34016523

RESUMEN

We propose that fructose-1,6-bisphosphate (F-1,6-BP) promotes a feedback loop between phosphofructokinase-1 (PFK1), phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt), and PFK2/PFKFB3, which enhances aerobic glycolysis and sustains effector T (Teff) cell activation, while oxidative metabolism is concomitantly downregulated. This regulation, promoted by low citrate and mitochondrial ATP synthesis, also sustains the Warburg effect in cancer cells.


Asunto(s)
Fructosadifosfatos/metabolismo , Glucólisis , Fosfofructoquinasa-1 , Linfocitos T , Adenosina Trifosfato/biosíntesis , Ácido Cítrico , Activación de Linfocitos , Mitocondrias , Neoplasias , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfofructoquinasa-1/genética , Fosfofructoquinasa-1/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Linfocitos T/metabolismo
13.
Cell Chem Biol ; 28(11): 1554-1568.e8, 2021 11 18.
Artículo en Inglés | MEDLINE | ID: mdl-33915105

RESUMEN

RNA-based sensors for intracellular metabolites are a promising solution to the emerging issue of metabolic heterogeneity. However, their development, i.e., the conversion of an aptamer into an in vivo-functional intracellular metabolite sensor, still harbors challenges. Here, we accomplished this for the glycolytic flux-signaling metabolite, fructose-1,6-bisphosphate (FBP). Starting from in vitro selection of an aptamer, we constructed device libraries with a hammerhead ribozyme as actuator. Using high-throughput screening in yeast with fluorescence-activated cell sorting (FACS), next-generation sequencing, and genetic-environmental perturbations to modulate the intracellular FBP levels, we identified a sensor that generates ratiometric fluorescent readout. An abrogated response in sensor mutants and occurrence of two sensor conformations-revealed by RNA structural probing-indicated in vivo riboswitching activity. Microscopy showed that the sensor can differentiate cells with different glycolytic fluxes within yeast populations, opening research avenues into metabolic heterogeneity. We demonstrate the possibility to generate RNA-based sensors for intracellular metabolites for which no natural metabolite-binding RNA element exits.


Asunto(s)
Técnicas Biosensibles , Fructosadifosfatos/química , ARN/análisis , Fructosadifosfatos/metabolismo , Glucólisis , ARN/metabolismo , Saccharomyces cerevisiae/metabolismo
14.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-33540748

RESUMEN

Tuberculosis (TB) remains one of the major health concerns worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While pfkB gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.


Asunto(s)
Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/enzimología , Fosfofructoquinasas/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Regulación Alostérica , Proteínas Bacterianas/antagonistas & inhibidores , Catálisis , Inducción Enzimática , Retroalimentación Fisiológica , Fructosadifosfatos/biosíntesis , Fructosadifosfatos/farmacología , Fructosafosfatos/metabolismo , Fructosafosfatos/farmacología , Gluconeogénesis , Glucólisis , Hexosafosfatos/metabolismo , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Cinética , L-Lactato Deshidrogenasa/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Oxígeno/farmacología , Fosfofructoquinasas/antagonistas & inhibidores , Piruvato Quinasa/metabolismo , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato
15.
Meat Sci ; 172: 108332, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33038798

RESUMEN

Phosphofructokinase-1 (PFK-1) is the most important enzyme controlling postmortem glycolysis in living skeletal muscle and is the most likely candidate for regulation of postmortem glycolysis. We investigated the regulation of PFK-1 activity by F-2, 6-BP and AMP under simulated postmortem conditions in porcine skeletal muscle. Six pigs were harvested and longissimus lumborum samples were collected immediately post-slaughter. PFK-1 activity was assayed using increasing concentrations of F-2, 6-BP or AMP, added to the buffer adjusted to different pH. Both F-2, 6-BP and AMP increased PFK-1 activity with increasing buffer pH from 5.5 to 7.0. A concentration of 50 µM F-2, 6-BP was required to increase PFK-1 activity which is very high compared to physiological concentration in the porcine skeletal muscle. However, physiological concentrations (50-150 µM) of AMP resulted in increased PFK-1 activity compared to 1-2 µM F-2, 6-BP. Thus, AMP may play a greater role in dictating the rate and extent of postmortem muscle glycolysis and pH decline as compared to F-2, 6-BP.


Asunto(s)
Adenosina Monofosfato/metabolismo , Fructosadifosfatos/metabolismo , Músculo Esquelético/enzimología , Fosfofructoquinasa-1/metabolismo , Animales , Activación Enzimática , Glucólisis , Concentración de Iones de Hidrógeno , Carne de Cerdo/análisis , Porcinos
16.
Front Endocrinol (Lausanne) ; 12: 797025, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-35095764

RESUMEN

Background: Glycolysis dysfunction is an important pathogenesis of podocyte injury in diabetic kidney disease (DKD). Foot process fusion of podocytes and increased albuminuria are markers of early DKD. Moreover, cytoskeletal remodeling has been found to be involved in the foot process fusion of podocytes. However, the connections between cytoskeletal remodeling and alterations of glycolysis in podocytes in DKD have not been clarified. Methods: mRNA sequencing of glomeruli obtained from db/db and db/m mice with albuminuria was performed to analyze the expression profiling of genes in glucose metabolism. Expressions of phosphofructokinase platelet type (PFKP) in the glomeruli of DKD patients were detected. Clotrimazole (CTZ) was used to explore the renal effects of PFKP inhibition in diabetic mice. Using Pfkp siRNA or recombinant plasmid to manipulate PFKP expression, the effects of PFKP on high glucose (HG) induced podocyte damage were assessed in vitro. The levels of fructose-1,6-bisphosphate (FBP) were measured. Targeted metabolomics was performed to observe the alterations of the metabolites in glucose metabolism after HG stimulation. Furthermore, aldolase type b (Aldob) siRNA or recombinant plasmid were applied to evaluate the influence of FBP level alteration on podocytes. FBP was directly added to podocyte culture media. Db/db mice were treated with FBP to investigate its effects on their kidney. Results: mRNA sequencing showed that glycolysis enzyme genes were altered, characterized by upregulation of upstream genes (Hk1, and Pfkp) and down-regulation of downstream genes of glycolysis (Pkm, and Ldha). Moreover, the expression of PFKP was increased in glomeruli of DKD patients. The CTZ group presented more severe renal damage. In vitro, the Pfkp siRNA group and ALDOB overexpression group showed much more induced cytoskeletal remodeling in podocytes, while overexpression of PFKP and suppression of ALDOB in vitro rescued podocytes from cytoskeletal remodeling through regulation of FBP levels and inhibition of the RhoA/ROCK1 pathway. Furthermore, targeted metabolomics showed FBP level was significantly increased in HG group compared with the control group. Exogenous FBP addition reduced podocyte cytoskeletal remodeling and renal damage of db/db mice. Conclusions: These findings provide evidence that PFKP may be a potential target for podocyte injury in DN and provide a rationale for applying podocyte glycolysis enhancing agents in patients with DKD.


Asunto(s)
Citoesqueleto/metabolismo , Nefropatías Diabéticas/genética , Fructosadifosfatos/metabolismo , Fosfofructoquinasa-1 Tipo C/genética , Podocitos/metabolismo , ARN Mensajero/metabolismo , Adulto , Albuminuria , Animales , Citoesqueleto/patología , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Femenino , Fructosa-Bifosfato Aldolasa/genética , Fructosa-Bifosfato Aldolasa/metabolismo , Glucólisis , Hexoquinasa/genética , Hexoquinasa/metabolismo , Humanos , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Fosfofructoquinasa-1 Tipo C/metabolismo , Podocitos/patología , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo
17.
J Appl Toxicol ; 41(7): 1050-1062, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33078453

RESUMEN

Fructose-1,6-bisphosphate (F1,6BP), an intermediate of the glycolytic pathway, has been found to play a promising anticancer effect; nevertheless, the mechanisms involved remain poorly understood. The present study aimed to evaluate the effect and mechanisms of F1,6BP in a human endometrial cancer cell line (Ishikawa). F1,6BP showed an antiproliferative and non-cytotoxic effect on endometrial cancer cells. These effects are related to the increase in reactive oxygen species (ROS) levels and mitochondrial membrane potential (ΔΨm). These harmful stimuli trigger the upregulation of the expression of pro-apoptotic genes (p53 and Bax), leading to the reduction of cell proliferation through inducing programmed cell death by apoptosis. Furthermore, F1,6BP-treated cells had the formation of autophagosomes induced, as well as a decrease in their proliferative capacity after withdrawing the treatment. Our results demonstrate that F1,6BP acts as an anticancer agent through the generation of mitochondrial instability, loss of cell function, and p53-dependent cell death. Thus, F1,6BP proves to be a potential molecule for use in the treatment against endometrial cancer.


Asunto(s)
Antineoplásicos/farmacología , Fructosadifosfatos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/genética , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Endometriales , Femenino , Fructosa/farmacología , Humanos , Mitocondrias/efectos de los fármacos
18.
Arch Biochem Biophys ; 695: 108633, 2020 11 30.
Artículo en Inglés | MEDLINE | ID: mdl-33075302

RESUMEN

A linked-function theory for allostery allows for a differentiation between those protein-ligand interactions that contribute the most to ligand binding and those protein-ligand interactions that contribute to the allosteric mechanism. This potential distinction is the basis for analogue studies used to determine which chemical moieties on the allosteric effector contribute to allostery. Although less recognized, the same separation of functions is possible for substrate-enzyme interactions. When evaluating allosteric regulation in human liver pyruvate kinase, the use of a range of monovalent cations (K+, NH4+, Rb+, Cs+, cyclohexylammonium+ and Tris+) altered substrate (phosphoenolpyruvate; PEP) affinity, but maintained similar allosteric responses to the allosteric activator, fructose-1,6-bisphosphate (Fru-1,6-BP). Because crystal structures indicate that the active site monovalent cation interacts directly with the phosphate moiety of the bound PEP substrate, we questioned if the phosphate moiety might contribute to substrate binding, but not to the allosteric mechanism. Here, we demonstrate that the binding of oxalate, a non-phosphorylated substrate/product analogue, is allosterically enhanced by Fru-1,6-BP. That observation is consistent with the concept that the phosphate moiety of PEP is not required for the allosteric function, even though that moiety likely contributes to determining substrate affinity.


Asunto(s)
Fructosadifosfatos/química , Hígado/enzimología , Fosfoenolpiruvato/química , Piruvato Quinasa/química , Regulación Alostérica , Fructosadifosfatos/metabolismo , Humanos , Fosfoenolpiruvato/metabolismo , Piruvato Quinasa/metabolismo
19.
mBio ; 11(5)2020 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-33109759

RESUMEN

Whereas the yeast Saccharomyces cerevisiae shows great preference for glucose as a carbon source, a deletion mutant in trehalose-6-phosphate synthase, tps1Δ, is highly sensitive to even a few millimolar glucose, which triggers apoptosis and cell death. Glucose addition to tps1Δ cells causes deregulation of glycolysis with hyperaccumulation of metabolites upstream and depletion downstream of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The apparent metabolic barrier at the level of GAPDH has been difficult to explain. We show that GAPDH isozyme deletion, especially Tdh3, further aggravates glucose sensitivity and metabolic deregulation of tps1Δ cells, but overexpression does not rescue glucose sensitivity. GAPDH has an unusually high pH optimum of 8.0 to 8.5, which is not altered by tps1Δ. Whereas glucose causes short, transient intracellular acidification in wild-type cells, in tps1Δ cells, it causes permanent intracellular acidification. The hxk2Δ and snf1Δ suppressors of tps1Δ restore the transient acidification. These results suggest that GAPDH activity in the tps1Δ mutant may be compromised by the persistently low intracellular pH. Addition of NH4Cl together with glucose at high extracellular pH to tps1Δ cells abolishes the pH drop and reduces glucose-6-phosphate (Glu6P) and fructose-1,6-bisphosphate (Fru1,6bisP) hyperaccumulation. It also reduces the glucose uptake rate, but a similar reduction in glucose uptake rate in a tps1Δ hxt2,4,5,6,7Δ strain does not prevent glucose sensitivity and Fru1,6bisP hyperaccumulation. Hence, our results suggest that the glucose-induced intracellular acidification in tps1Δ cells may explain, at least in part, the apparent glycolytic bottleneck at GAPDH but does not appear to fully explain the extreme glucose sensitivity of the tps1Δ mutant.IMPORTANCE Glucose catabolism is the backbone of metabolism in most organisms. In spite of numerous studies and extensive knowledge, major controls on glycolysis and its connections to the other metabolic pathways remain to be discovered. A striking example is provided by the extreme glucose sensitivity of the yeast tps1Δ mutant, which undergoes apoptosis in the presence of just a few millimolar glucose. Previous work has shown that the conspicuous glucose-induced hyperaccumulation of the glycolytic metabolite fructose-1,6-bisphosphate (Fru1,6bisP) in tps1Δ cells triggers apoptosis through activation of the Ras-cAMP-protein kinase A (PKA) signaling pathway. However, the molecular cause of this Fru1,6bisP hyperaccumulation has remained unclear. We now provide evidence that the persistent drop in intracellular pH upon glucose addition to tps1Δ cells likely compromises the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a major glycolytic enzyme downstream of Fru1,6bisP, due to its unusually high pH optimum. Our work highlights the potential importance of intracellular pH fluctuations for control of major metabolic pathways.


Asunto(s)
Glucosa/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Saccharomyces cerevisiae/enzimología , Apoptosis , Citoplasma/química , Fermentación , Fructosadifosfatos/análisis , Eliminación de Gen , Glucosa-6-Fosfato/análisis , Glucólisis , Concentración de Iones de Hidrógeno , Redes y Vías Metabólicas , Mutación , Saccharomyces cerevisiae/genética
20.
Cell Transplant ; 29: 963689720950226, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32841050

RESUMEN

Apoptosis is a vital pathological factor that accounts for the poor prognosis of traumatic spinal cord injury (t-SCI). The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) is a critical regulator for energy metabolism and proven to have antiapoptotic effects. This study aimed to investigate the neuroprotective role of PFKFB3 in t-SCI. A compressive clip was introduced to establish the t-SCI model. Herein, we identified that PFKFB3 was extensively distributed in neurons, and PFKFB3 levels significantly increased and peaked 24 h after t-SCI. Additionally, knockdown of PFKFB3 inhibited glycolysis, accompanied by aggravated neuronal apoptosis and white matter injury, while pharmacological activation of PFKFB3 with meclizine significantly enhanced glycolysis, attenuated t-SCI-induced spinal cord injury, and alleviated neurological impairment. The PFKFB3 agonist, meclizine, activated cyclin-dependent kinase 1 (CDK1) and promoted the phosphorylation of p27, ultimately suppressing neuronal apoptosis. However, the neuroprotective effects of meclizine against t-SCI were abolished by the CDK1 antagonist, RO3306. In summary, our data demonstrated that PFKFB3 contributes robust neuroprotection against t-SCI by enhancing glycolysis and modulating CDK1-related antiapoptotic signals. Moreover, targeting PFKFB3 may be a novel and promising therapeutic strategy for t-SCI.


Asunto(s)
Apoptosis , Proteína Quinasa CDC2/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Glucólisis , Neuronas/patología , Fosfofructoquinasa-2/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Animales , Apoptosis/efectos de los fármacos , Fructosadifosfatos/metabolismo , Técnicas de Silenciamiento del Gen , Glucólisis/efectos de los fármacos , Ácido Láctico/metabolismo , Masculino , Meclizina/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Modelos Biológicos , Actividad Motora/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosforilación/efectos de los fármacos , Quinolinas/farmacología , Ratas Sprague-Dawley , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Médula Espinal/fisiopatología , Médula Espinal/ultraestructura , Traumatismos de la Médula Espinal/fisiopatología , Tiazoles/farmacología , Factores de Tiempo , Regulación hacia Arriba/efectos de los fármacos , Sustancia Blanca/lesiones , Sustancia Blanca/patología
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