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Archives of Biochemistry and Biophysics Jul 2023The phosphofructokinase (Pfk) reaction represents one of the key regulatory points in glycolysis. While most organisms encode for Pfks that use ATP as phosphoryl donor,...
The phosphofructokinase (Pfk) reaction represents one of the key regulatory points in glycolysis. While most organisms encode for Pfks that use ATP as phosphoryl donor, some organisms also encode for PP-dependent Pfks. Despite this central role, the biochemical characteristics as well as the physiological role of both Pfks is often not known. Clostridium thermocellum is an example of a microorganism that encodes for both Pfks, however, only PP-Pfk activity has been detected in cell-free extracts and little is known about the regulation and function of both enzymes. In this study, the ATP- and PP-Pfk of C. thermocellum were purified and biochemically characterized. No allosteric regulators were found for PP-Pfk amongst common effectors. With fructose-6-P, PP, fructose-1,6-bisP, and P PP-Pfk showed high specificity (K < 0.62 mM) and maximum activity (V > 156 U mg). In contrast, ATP-Pfk showed much lower affinity (K of 9.26 mM) and maximum activity (14.5 U mg) with fructose-6-P. In addition to ATP, also GTP, UTP and ITP could be used as phosphoryl donors. The catalytic efficiency with GTP was 7-fold higher than with ATP, suggesting that GTP is the preferred substrate. The enzyme was activated by NH, and pronounced inhibition was observed with GDP, FBP, PEP, and especially with PP (K of 0.007 mM). Characterization of purified ATP-Pfks originating from eleven different bacteria, encoding for only ATP-Pfk or for both ATP- and PP-Pfk, identified that PP inhibition of ATP-Pfks could be a common phenomenon for organisms with a PP-dependent glycolysis.
Topics: Phosphofructokinases; Clostridium thermocellum; Diphosphates; Amino Acid Sequence; Phosphofructokinase-1; Bacteria; Adenosine Triphosphate; Guanosine Triphosphate; Kinetics
PubMed: 37380119
DOI: 10.1016/j.abb.2023.109676 -
Reproduction in Domestic Animals =... Oct 2017The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar...
The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well-known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential-interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/10 spermatozoa, the activity of NAD- and NADP-dependent IDH was 0.111 ± 0.005 U/10 and 2.22 ± 0.14 U/10 spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/10 spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa.
Topics: Acrosome Reaction; Animals; Bicarbonates; Female; Follicular Fluid; Isocitrate Dehydrogenase; Malate Dehydrogenase; Male; Phosphofructokinases; Sperm Capacitation; Sperm Motility; Spermatozoa; Sus scrofa; Tartronates
PubMed: 28397297
DOI: 10.1111/rda.12973 -
Cancer Medicine Jul 2023Patients with cervical cancer (CC) may experience local recurrence very often after treatment; when only clinical parameters are used, most cases are diagnosed in late...
The mRNA and protein levels of the glycolytic enzymes lactate dehydrogenase A (LDHA) and phosphofructokinase platelet (PFKP) are good predictors of survival time, recurrence, and risk of death in cervical cancer patients.
INTRODUCTION
Patients with cervical cancer (CC) may experience local recurrence very often after treatment; when only clinical parameters are used, most cases are diagnosed in late stages, which decreases the chance of recovery. Molecular markers can improve the prediction of clinical outcome. Glycolysis is altered in 70% of CCs, so molecular markers of this pathway associated with the aggressiveness of CC can be identified.
METHODS
The expression of 14 glycolytic genes was analyzed in 97 CC and 29 healthy cervical tissue (HCT) with microarray; only LDHA and PFKP were validated at the mRNA and protein levels in 36 of those CC samples and in 109 new CC samples, and 31 HCT samples by qRT-PCR, Western blotting, or immunohistochemistry. A replica analysis was performed on 295 CC from The Cancer Genome Atlas (TCGA) database.
RESULTS
The protein expression of LDHA and PFKP was associated with poor overall survival [OS: LDHA HR = 4.0 (95% CI = 1.4-11.1); p = 8.0 × 10 ; PFKP HR = 3.3 (95% CI = 1.1-10.5); p = 4.0 × 10 ] and disease-free survival [DFS: LDHA HR = 4.5 (95% CI = 1.9-10.8); p = 1.0 × 10 ; PFKP HR = 3.2 (95% CI = 1.2-8.2); p = 1.8 × 10 ] independent of FIGO clinical stage, and the results for mRNA expression were similar. The risk of death was greater in patients with overexpression of both biomarkers than in patients with advanced FIGO stage [HR = 8.1 (95% CI = 2.6-26.1; p = 4.3 × 10 ) versus HR = 7 (95% CI 1.6-31.1, p = 1.0 × 10 )] and increased exponentially as the expression of LDHA and PFKP increased.
CONCLUSIONS
LDHA and PFKP overexpression at the mRNA and protein levels was associated with poor OS and DFS and increased risk of death in CC patients regardless of FIGO stage. The measurement of these two markers could be very useful for evaluating clinical evolution and the risk of death from CC and could facilitate better treatment decision making.
Topics: Female; Humans; Biomarkers; Glycolysis; L-Lactate Dehydrogenase; Lactate Dehydrogenase 5; Phosphofructokinases; RNA, Messenger; Uterine Cervical Neoplasms
PubMed: 37326348
DOI: 10.1002/cam4.6123 -
Reproduction in Domestic Animals =... Dec 2014Oocyte maturation depends on the metabolic activity of cumulus-oocyte complex (COC) that performs nutritive and regulatory functions during this process. In this work,...
Oocyte maturation depends on the metabolic activity of cumulus-oocyte complex (COC) that performs nutritive and regulatory functions during this process. In this work, the enzymes [phosphofructokinase (PFK) and malate dehydrogenase (MDH)] were tested to elucidate the metabolic profile of porcine COCs during the in vitro maturation (IVM). Enzymatic activity was expressed in U/COC and U/mg protein (specific activity) as mean ± SEM. In vitro maturation was performed with 2-oxoglutarate (5, 10 and 20 mm) or hydroxymalonate (30, 60 and 100 mm) inhibitors of PFK and MDH, respectively. The PFK and MDH activities (U) remained constant during maturation. For PFK, the U were (2.48 ± 0.23) 10(-5) and (2.54 ± 0.32) 10(-5) , and for MDH, the U were (4.72 ± 0.42) 10(-5) and (4.38 ± 0.25) 10(-5) for immature and in vitro matured COCs, respectively. The specific activities were significantly lower after IVM, for PFK (4.29 ± 0.48) 10(-3) and (0.94 ± 0.12) 10(-3) , and for MDH (9.08 ± 0.93) 10(-3) and (1.89 ± 0.10) 10(-3) for immature and in vitro matured COCs, respectively. In vitro maturation percentages and enzymatic activity diminished with 20 mm 2-oxoglutarate or 60 mm hydroxymalonate (p < 0.05). Viability was not affected by any concentration of the inhibitors evaluated. The U remained unchanged during IVM; however, the increase in the total protein content per COC provoked a decrease in the specific activity of both enzymes. Phosphofructokinase and MDH necessary for oocyte IVM would be already present in the immature oocyte. The presence of inhibitors of these enzymes impairs the meiotic maturation. Therefore, the participation of these enzymes in the energy metabolism of the porcine oocyte during IVM is confirmed in this study.
Topics: Animals; Cell Survival; Cumulus Cells; Gene Expression Regulation, Enzymologic; In Vitro Oocyte Maturation Techniques; Ketoglutaric Acids; Malate Dehydrogenase; Meiosis; Oocytes; Phosphofructokinases; Swine; Tartronates
PubMed: 25307885
DOI: 10.1111/rda.12437 -
The Journal of Cell Biology Aug 2017Despite abundant knowledge of the regulation and biochemistry of glycolytic enzymes, we have limited understanding on how they are spatially organized in the cell.... (Comparative Study)
Comparative Study
Despite abundant knowledge of the regulation and biochemistry of glycolytic enzymes, we have limited understanding on how they are spatially organized in the cell. Emerging evidence indicates that nonglycolytic metabolic enzymes regulating diverse pathways can assemble into polymers. We now show tetramer- and substrate-dependent filament assembly by phosphofructokinase-1 (PFK1), which is considered the "gatekeeper" of glycolysis because it catalyzes the step committing glucose to breakdown. Recombinant liver PFK1 (PFKL) isoform, but not platelet PFK1 (PFKP) or muscle PFK1 (PFKM) isoforms, assembles into filaments. Negative-stain electron micrographs reveal that filaments are apolar and made of stacked tetramers oriented with exposed catalytic sites positioned along the edge of the polymer. Electron micrographs and biochemical data with a PFKL/PFKP chimera indicate that the PFKL regulatory domain mediates filament assembly. Quantified live-cell imaging shows dynamic properties of localized PFKL puncta that are enriched at the plasma membrane. These findings reveal a new behavior of a key glycolytic enzyme with insights on spatial organization and isoform-specific glucose metabolism in cells.
Topics: Blood Platelets; Cell Membrane; Glucose; Glycolysis; HEK293 Cells; Humans; Kinetics; Liver; Microscopy, Confocal; Microscopy, Electron, Transmission; Microscopy, Video; Muscle, Skeletal; Phosphofructokinase-1, Liver Type; Phosphofructokinase-1, Muscle Type; Phosphofructokinase-1, Type C; Protein Multimerization; Protein Structure, Quaternary; Recombinant Proteins; Structure-Activity Relationship; Substrate Specificity; Time-Lapse Imaging
PubMed: 28646105
DOI: 10.1083/jcb.201701084 -
The Journal of Cell Biology Jul 2022In this issue, Harris et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202203095) show that phosphofructokinase is a substrate for ubiquitination by Fbxo7, a key...
In this issue, Harris et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202203095) show that phosphofructokinase is a substrate for ubiquitination by Fbxo7, a key protein in the ubiquitination pathway. Their findings point to a new interplay between metabolic enzyme degradation in the regulation of T cells.
Topics: F-Box Proteins; Humans; Phosphofructokinases; T-Lymphocytes; Ubiquitination
PubMed: 35695892
DOI: 10.1083/jcb.202206006 -
Journal of Cancer Research and Clinical... Sep 2023This study was to investigate the biological effect of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2) in colorectal cancer (CRC).
PURPOSE
This study was to investigate the biological effect of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2) in colorectal cancer (CRC).
METHODS
PFKFB2 was selected by metabolism polymerase chain reaction (PCR) array from CRC cells under alkaline culture medium (pH 7.4) and acidic culture medium (pH 6.8). The expression of PFKFB2 mRNA and protein was detected by quantitative real-time PCR and immunohistochemistry in 70 paired fresh and 268 paired paraffin-embedded human CRC tissues, respectively, and then the prognostic value of PFKFB2 was investigated. The effects of PFKFB2 on CRC cells were also verified in vitro, which were through detecting the change of migration, invasion, sphere formation, proliferation, colony formation, and extracellular acidification rate of CRC cells after PFKFB2 knockdown in alkaline culture medium (pH 7.4) and overexpression in acidic culture medium (pH 6.8).
RESULTS
PFKFB2 expression was downregulated in acidic culture medium (pH 6.8). In addition, we found PFKFB2 expression decreased in human CRC tissues compared with the adjacent normal tissues. Furthermore, the OS and DFS rate of CRC patients with low PFKFB2 expression was significantly shorter than those of patients with high PFKFB2 expression. Multivariate analysis indicated that low PFKFB2 expression was an independent prognostic factor for both OS and DFS in CRC patients. Moreover, the abilities of migration, invasion, spheroidizing ability, proliferation, and colony formation of CRC cells were significantly increased after depletion of PFKFB2 in alkaline culture medium (pH 7.4) and decreased after overexpression of PFKFB2 in acidic culture medium (pH 6.8) in vitro. Epithelial-mesenchymal transition (EMT) pathway was found and verified involved in the PFKFB2-mediated regulation of metastatic function in CRC cells. Further, glycolysis of CRC cells was significantly elevated after knockdown of PFKFB2 in alkaline culture medium (pH 7.4) and decreased after overexpression of PFKFB2 in acidic culture medium (pH 6.8).
CONCLUSION
PFKFB2 expression is downregulated in CRC tissues and associated with worse survival for CRC patients. PFKFB2 could inhibit metastasis and the malignant progression of CRC cells by suppressing EMT and glycolysis.
Topics: Humans; Biomarkers; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Glycolysis; Phosphofructokinase-2; Prognosis
PubMed: 37311985
DOI: 10.1007/s00432-023-04946-1 -
Biomedicine & Pharmacotherapy =... Nov 2022Nonalcoholic fatty liver disease (NAFLD), often associated with obesity, is becoming one of the most common liver diseases worldwide. It is estimated to affect one...
Nonalcoholic fatty liver disease (NAFLD), often associated with obesity, is becoming one of the most common liver diseases worldwide. It is estimated to affect one billion individuals and may be present in approximately 25% of the population globally. NAFLD is viewed as a hepatic manifestation of metabolic syndrome, with humans and animal models presenting dyslipidemia, hypertension, and diabetes. The gut-liver axis has been considered the main pathogenesis branch for NAFLD development. Considering that foods or beverages could modulate the gastrointestinal tract, immune system, energy homeostasis regulation, and even the gut-liver axis, we conducted an exploratory study to analyze the effects of kombucha probiotic on hepatic steatosis, glucose tolerance, and hepatic enzymes involved in carbohydrate and fat metabolism using a pre-clinical model. The diet-induced obese mice presented glucose intolerance, hyperinsulinemia, hepatic steatosis, increased collagen fiber deposition in liver vascular spaces, and upregulated TNF-alpha and SREBP-1 gene expression. Mice receiving the kombucha supplement displayed improved glucose tolerance, reduced hyperinsulinemia, decreased citrate synthase and phosphofructokinase-1 enzyme activities, downregulated G-protein-coupled bile acid receptor, also known as TGR5, and farnesol X receptor gene expression, and attenuated steatosis and hepatic collagen fiber deposition. The improvement in glucose tolerance was accompanied by the recovery of acute insulin-induced liver AKT serine phosphorylation. Thus, it is possible to conclude that this probiotic drink has a beneficial effect in reducing the metabolic alterations associated with diet-induced obesity. This probiotic beverage deserves an extension of studies to confirm or refute its potentially beneficial effects.
Topics: Humans; Mice; Animals; Mice, Obese; Non-alcoholic Fatty Liver Disease; Kombucha Tea; Sterol Regulatory Element Binding Protein 1; Tumor Necrosis Factor-alpha; Citrate (si)-Synthase; Farnesol; Proto-Oncogene Proteins c-akt; Liver; Obesity; Insulin Resistance; Insulin; Glucose; Bile Acids and Salts; Carbohydrates; Serine; Phosphofructokinase-1; GTP-Binding Proteins; Collagen; Mice, Inbred C57BL; Diet, High-Fat
PubMed: 36095960
DOI: 10.1016/j.biopha.2022.113660 -
Carcinogenesis Aug 2022T lymphoma invasion and metastasis 1 (Tiam1) as a tumor-associated gene specifically activates Rho-like GTPases Rac1 and implicates in the invasive phenotype of many...
T lymphoma invasion and metastasis 1 (Tiam1) as a tumor-associated gene specifically activates Rho-like GTPases Rac1 and implicates in the invasive phenotype of many cancers. Altering the glycolytic pathway is foreseen as a sound approach to trigger cancer regression. However, the mechanism of Tiam1 in breast cancer (BC) glycolysis reprogramming remains to be clarified. Here, we reported the Tiam1 high expression and prognostic significance in BC. In vitro and in vivo experimental assays identified the functional role of Tiam1 in promoting BC cell proliferation, metastasis and glycolysis reprogramming. Mechanistically, we showed for the first time that Tiam1 could interact with the crucial glycolytic enzyme phosphofructokinase, liver type (PFKL) and promote the evolution of BC in a PFKL-dependent manner. Moreover, miR-21-5p was found to exacerbate the BC proliferation and aggression by targeting Tiam1. Altogether, our study highlights the critical role of Tiam1 in BC development and that the miR-21-5p/Tiam1/PFKL signaling pathway may serve as a target for new anti-BC therapeutic strategies.
Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Glycolysis; Humans; Liver; MicroRNAs; Neoplasm Invasiveness; Phosphofructokinase-1, Liver Type; Phosphofructokinases; T-Lymphoma Invasion and Metastasis-inducing Protein 1
PubMed: 35511493
DOI: 10.1093/carcin/bgac039 -
Applied Microbiology and Biotechnology May 2017D-Ribulose-5-phosphate-3-epimerase (RPE) and 6-phosphofructokinase (PFK) catalyse two reactions in the ribulose monophosphate (RuMP) cycle in Bacillus methanolicus. The...
D-Ribulose-5-phosphate-3-epimerase (RPE) and 6-phosphofructokinase (PFK) catalyse two reactions in the ribulose monophosphate (RuMP) cycle in Bacillus methanolicus. The B. methanolicus wild-type strain MGA3 possesses two putative rpe and pfk genes encoded on plasmid pBM19 (rpe1-MGA3 and pfk1-MGA3) and on the chromosome (rpe2-MGA3 and pfk2-MGA3). The wild-type strain PB1 also encodes putative rpe and pfk genes on plasmid pBM20 (rpe1-PB1 and pfk1-PB1*); however, it only harbours a chromosomal pfk gene (pfk2-PB1). Transcription of the plasmid-encoded genes was 10-fold to 15-fold upregulated in cells growing on methanol compared to mannitol, while the chromosomal genes were transcribed at similar levels under both conditions in both strains. All seven gene products were recombinantly produced in Escherichia coli, purified and biochemically characterized. All three RPEs were active as hexamers, catalytically stimulated by Mg and Mn and displayed similar K' values (56-75 μM) for ribulose 5-phosphate. Rpe2-MGA3 showed displayed 2-fold lower V (49 U/mg) and a significantly reduced thermostability compared to the two Rpe1 proteins. Pfk1-PB1* was shown to be non-functional. The PFKs were active both as octamers and as tetramers, were catalytically stimulated by Mg and Mn, and displayed similar thermostabilities. The PFKs have similar K values for fructose 6-phosphate (0.61-0.94 μM) and for ATP (0.38-0.82 μM), while Pfk1-MGA3 had a 2-fold lower V (6.3 U/mg) compared to the two Pfk2 proteins. Our results demonstrate that MGA3 and PB1 exert alternative solutions to plasmid-dependent methylotrophy, including genetic organization, regulation, and biochemistry of RuMP cycle enzymes.
Topics: Bacillus; Bacterial Proteins; Carbohydrate Epimerases; Chromosomes, Bacterial; Escherichia coli; Kinetics; Mannitol; Metabolic Networks and Pathways; Methanol; Phosphofructokinase-1; Plasmids; Recombinant Proteins; Ribulosephosphates; Sequence Analysis, DNA
PubMed: 28213736
DOI: 10.1007/s00253-017-8173-0