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Science Advances Mar 2023Isoenzyme divergence is a prevalent mechanism governing tissue-specific and developmental stage-specific metabolism in mammals. The lactate dehydrogenase (LDH) isoenzyme...
Isoenzyme divergence is a prevalent mechanism governing tissue-specific and developmental stage-specific metabolism in mammals. The lactate dehydrogenase (LDH) isoenzyme spectrum reflects the tissue-specific metabolic status. We found that three tetrameric isoenzymes composed of LDHA and LDHB (LDH-3/4/5) comprise the LDH spectrum in T cells. Genetically deleting or altered the isoenzyme spectrum by removing all heterotetramers and leaving T cells with LDH-1 (the homotetramer of LDHB) or LDH-5 (the homotetramer of LDHA), respectively. Accordingly, deleting suppressed glycolysis, cell proliferation, and differentiation. Unexpectedly, deleting enhanced glycolysis but suppressed T cell differentiation, indicating that an optimal zone of glycolytic activity is required to maintain cell fitness. Mechanistically, the LDH isoenzyme spectrum imposed by LDHA and LDHB is necessary to optimize glycolysis to maintain a balanced nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide hydrogen pool. Our results suggest that the LDH isoenzyme spectrum enables "Goldilocks levels" of glycolytic and redox activity to control T cell differentiation.
Topics: Animals; Isoenzymes; NAD; T-Lymphocytes; L-Lactate Dehydrogenase; Lactate Dehydrogenase 5; Glycolysis; Cell Differentiation; Mammals
PubMed: 36961904
DOI: 10.1126/sciadv.add9554 -
Clinical Biochemistry May 2021Lactate dehydrogenase (LDH) is a key enzyme that functions as a marker of cell damage. Its activity can be measured by a variety of laboratory methods. To eliminate...
BACKGROUND
Lactate dehydrogenase (LDH) is a key enzyme that functions as a marker of cell damage. Its activity can be measured by a variety of laboratory methods. To eliminate inter-method bias and enhance equivalence among different measurement procedures, LDH detection needs to be standardized.
METHODS
Optimized sequences coding for lactate dehydrogenase subunit A (LDH-A) and subunit B (LDH-B) were synthesized and cloned into the pRSFDuet vector, and then the constructed 6His-LDHA-pRSFDuet, 6His-LDHB-pRSFDuet, and 6His-LDHA-LDHB-pRSFDuet plasmids were transformed into Escherichia coli BL21 (DE3) for expression. The enzyme activity and specific activity of recombinant LDHs were detected. Electrophoresis of LDH isoenzymes was performed. The stability of recombinant LDHs and serum LDH was evaluated. Commutability of recombinant LDH-B was studied by the IFCC reference method and six routine methods.
RESULTS
Three plasmids were constructed and three highly concentrated recombinant LDH isoenzymes were obtained. The specific activities of LDH-A, LDH-AB, and LDH-B were 18.08 U/mg, 21.74 U/mg, and 14.18 U/mg, respectively. There was a desirable linear correlation between the activities of recombinant LDH isoenzymes and their protein concentrations. Electrophoresis of LDH isoenzymes showed that the recombinant LDH-B corresponded to LDH1 and it demonstrated good stability at 4 °C and 25 °C for 5 weeks. LDH-B formulations in saline-bovine serum albumin solution and human serum matrix were commutable for six routine methods.
CONCLUSION
Human recombinant LDH-B has great potential to become an improved and less expensive standard or reference material in external quality assessment for clinical LDH measurement.
Topics: Clinical Enzyme Tests; Humans; L-Lactate Dehydrogenase; Lactate Dehydrogenases; Recombinant Proteins; Reference Standards
PubMed: 33617848
DOI: 10.1016/j.clinbiochem.2021.02.002 -
ELife Jun 2022Lactate oxidation with NAD as electron acceptor is a highly endergonic reaction. Some anaerobic bacteria overcome the energetic hurdle by flavin-based electron...
Lactate oxidation with NAD as electron acceptor is a highly endergonic reaction. Some anaerobic bacteria overcome the energetic hurdle by flavin-based electron bifurcation/confurcation (FBEB/FBEC) using a lactate dehydrogenase (Ldh) in concert with the electron-transferring proteins EtfA and EtfB. The electron cryo-microscopically characterized (Ldh-EtfAB) complex of at 2.43 Å resolution consists of a mobile EtfAB shuttle domain located between the rigid central Ldh and the peripheral EtfAB base units. The FADs of Ldh and the EtfAB shuttle domain contact each other thereby forming the D (dehydrogenation-connected) state. The intermediary Glu37 and Glu139 may harmonize the redox potentials between the FADs and the pyruvate/lactate pair crucial for FBEC. By integrating Alphafold2 calculations a plausible novel B (bifurcation-connected) state was obtained allowing electron transfer between the EtfAB base and shuttle FADs. Kinetic analysis of enzyme variants suggests a correlation between NAD binding site and D-to-B-state transition implicating a 75° rotation of the EtfAB shuttle domain. The FBEC inactivity when truncating the ferredoxin domain of EtfA substantiates its role as redox relay. Lactate oxidation in Ldh is assisted by the catalytic base His423 and a metal center. On this basis, a comprehensive catalytic mechanism of the FBEC process was proposed.
Topics: Electron Transport; Electrons; Kinetics; L-Lactate Dehydrogenase; Lactates; NAD; Oxidation-Reduction
PubMed: 35748623
DOI: 10.7554/eLife.77095 -
World Journal of Microbiology &... Nov 2020Lactate are proved to be attractive electron donor for the production of n-caproic acid (CA) that is a high value-added fuel precursor and chemical feedstock, but little...
Lactate are proved to be attractive electron donor for the production of n-caproic acid (CA) that is a high value-added fuel precursor and chemical feedstock, but little is known about molecular mechanism of lactate transformation. In the present study, the gene for L-lactate dehydrogenase (LDH, EC.1.1.1.27) from a Ruminococcaceae strain CPB6 was cloned and expressed in Escherichia coli BL21 (DE3) with plasmid pET28a. The recombinant LDH exhibited molecular weight of 36-38 kDa in SDS-PAGE. The purified LDH was found to have the maximal oxidation activity of 29.6 U/mg from lactate to pyruvate at pH 6.5, and the maximal reduction activity of 10.4 U/mg from pyruvate to lactate at pH 8.5, respectively. Strikingly, its oxidative activity predominates over reductive activity, leading to a 17-fold increase for the utilization of lactate in E. coli/pET28a-LDH than E. coli/pET28a. The CPB6 LDH gene encodes a 315 amino acid protein sharing 42.19% similarity with Clostridium beijerinckii LDH, and lower similarity with LDHs of other organisms. Significant difference were observed between the CPB6 LDH and C. beijerinckii and C. acetobutylicum LDH in the predicted tertiary structure and active center. Further, X-ray crystal structure analysis need to be performed to verify the specific active center of the CPB6 LDH and its role in the conversion of lactate into CA.
Topics: Bacterial Proteins; Cloning, Molecular; Clostridiales; Crystallography, X-Ray; Escherichia coli; Hydrogen-Ion Concentration; L-Lactate Dehydrogenase; Lactic Acid; Models, Molecular; Molecular Weight; Plasmids; Protein Structure, Tertiary; Pyruvic Acid; Recombinant Proteins
PubMed: 33170386
DOI: 10.1007/s11274-020-02958-4 -
Acta Oncologica (Stockholm, Sweden) Jul 2015In cancer cells, metabolism is shifted to aerobic glycolysis with lactate production coupled with a higher uptake of glucose as the main energy source. Lactate... (Meta-Analysis)
Meta-Analysis Review
BACKGROUND
In cancer cells, metabolism is shifted to aerobic glycolysis with lactate production coupled with a higher uptake of glucose as the main energy source. Lactate dehydrogenase (LDH) catalyzes the reduction of pyruvate to form lactate, and serum level is often raised in aggressive cancer and hematological malignancies. We have assessed the prognostic value of LDH in solid tumors.
MATERIAL AND METHODS
A systematic review of electronic databases was conducted to identify publications exploring the association of LDH with clinical outcome in solid tumors. Overall survival (OS) was the primary outcome, and cancer-specific survival (CSS), progression-free survival (PFS), and disease-free survival (DFS) were secondary outcomes. Data from studies reporting a hazard ratio (HR) and 95% confidence interval (CI) were pooled in a meta-analysis. Pooled HRs were computed and weighted using generic inverse-variance and random-effect modeling. All statistical tests were two-sided.
RESULTS
Seventy-six studies comprising 22 882 patients, mainly with advanced disease, were included in the analysis. Median cut-off of serum LDH was 245 U/L. Overall, higher LDH levels were associated with a HR for OS of 1.7 (95% CI 1.62-1.79; p < 0.00001) in 73 studies. The prognostic effect was highest in renal cell, melanoma, gastric, prostate, nasopharyngeal and lung cancers (all p < 0.00001). HRs for PFS was 1.75 (all p < 0.0001).
CONCLUSIONS
A high serum LDH level is associated with a poor survival in solid tumors, in particular melanoma, prostate and renal cell carcinomas, and can be used as a useful and inexpensive prognostic biomarker in metastatic carcinomas.
Topics: Humans; L-Lactate Dehydrogenase; Neoplasms; Prognosis
PubMed: 25984930
DOI: 10.3109/0284186X.2015.1043026 -
Oncoimmunology 2020Lactate dehydrogenase (LDH) levels are inversely related with response to checkpoint inhibitors. Elevated LDH levels are the product of enhanced glycolytic activity of... (Review)
Review
Lactate dehydrogenase (LDH) levels are inversely related with response to checkpoint inhibitors. Elevated LDH levels are the product of enhanced glycolytic activity of the tumor and tumor necrosis due to hypoxia, the latter being associated with high tumor burden. In this review, we elucidate the effects of glycolysis and hypoxia on antitumor immunity and set forth ways to improve response to immunotherapy in cancer patients with elevated LDH levels. We discuss the current knowledge on combining immunotherapy with glycolysis inhibitors, anti-acidifying drugs, anti-angiogenic or cytoreductive therapy.
Topics: Biomarkers; Glycolysis; Humans; Immunotherapy; L-Lactate Dehydrogenase; Neoplasms
PubMed: 32158624
DOI: 10.1080/2162402X.2020.1731942 -
Biotechnology and Applied Biochemistry Dec 2022This study presents that covalent immobilization technique has been utilized for the immobilization of l-lactate dehydrogenase (l-LDH) from porcine on mesoporous silica....
This study presents that covalent immobilization technique has been utilized for the immobilization of l-lactate dehydrogenase (l-LDH) from porcine on mesoporous silica. To develop mesoporous silica as support material for use in l-LDH immobilization, the particle surfaces were functionalized with 3-aminopropyltrimethoxysilane and further conjugated with glutaraldehyde. The effect of some parameters such as glutaraldehyde concentration, immobilization pH, initial enzyme concentration, and immobilization time was investigated and the optimum conditions for these parameters were determined as 1% (w/v), pH 8.0, 1 mg/ml, and 120 min, respectively. The maximum working pH and temperature for the oxidation of lactate to pyruvate reaction were determined as 10.0 and 35°C for free and 9.0 and 40°C for immobilized l-LDH, respectively. The kinetic parameters (K and V ) of l-LDH for the oxidation of lactate to pyruvate reaction were examined as 1.02 mM and 7.58 U/mg protein for free and 0.635 mM and 1.7 U/mg protein for immobilized l-LDH, respectively. Moreover, the immobilized l-LDH was 1.3-fold more stable than free l-LDH at 25°C according to calculated t values. The immobilized l-LDH retained 80% of its initial activity in a batch reactor after 14 reuses.
Topics: Swine; Animals; Enzymes, Immobilized; Enzyme Stability; L-Lactate Dehydrogenase; Silicon Dioxide; Glutaral; Hydrogen-Ion Concentration; Temperature; Lactates; Kinetics
PubMed: 34962677
DOI: 10.1002/bab.2304 -
Cancer Letters Apr 2024Lactate dehydrogenase A (LDHA) serves as a key regulator of the Warburg Effect by catalyzing the conversion of pyruvate to lactate in the final step of glycolysis. Both...
Lactate dehydrogenase A (LDHA) serves as a key regulator of the Warburg Effect by catalyzing the conversion of pyruvate to lactate in the final step of glycolysis. Both the expression level and enzyme activity of LDHA are upregulated in cancers, however, the underlying mechanism remains incompletely understood. Here, we show that LDHA is post-translationally palmitoylated by ZDHHC9 at cysteine 163, which promotes its enzyme activity, lactate production, and reduces reactive oxygen species (ROS) generation. Replacement of endogenous LDHA with a palmitoylation-deficient mutant leads to reduced pancreatic cancer cell proliferation, increased T-cell infiltration, and limited tumor growth; it also affects pancreatic cancer cell response to chemotherapy. Moreover, LDHA palmitoylation is upregulated in gemcitabine resistant pancreatic cancer cells. Clinically, ZDHHC9 is upregulated in pancreatic cancer and correlated with poor prognoses for patients. Overall, our findings identify ZDHHC9-mediated palmitoylation as a positive regulator of LDHA, with potentially significant implications for cancer etiology and targeted therapy for pancreatic cancer.
Topics: Humans; L-Lactate Dehydrogenase; Lipoylation; Cell Line, Tumor; Lactate Dehydrogenase 5; Pancreatic Neoplasms; Glycolysis; Cell Proliferation; Lactates
PubMed: 38331089
DOI: 10.1016/j.canlet.2024.216696 -
Aging Feb 2023The astrocyte-neuron lactate shuttle hypothesis posits that glial-generated lactate is transported to neurons to fuel metabolic processes required for long-term memory....
The astrocyte-neuron lactate shuttle hypothesis posits that glial-generated lactate is transported to neurons to fuel metabolic processes required for long-term memory. Although studies in vertebrates have revealed that lactate shuttling is important for cognitive function, it is uncertain if this form of metabolic coupling is conserved in invertebrates or is influenced by age. Lactate dehydrogenase (Ldh) is a rate limiting enzyme that interconverts lactate and pyruvate. Here we genetically manipulated expression of lactate dehydrogenase (dLdh) in neurons or glia to assess the impact of altered lactate metabolism on invertebrate aging and long-term courtship memory at different ages. We also assessed survival, negative geotaxis, brain neutral lipids (the core component of lipid droplets) and brain metabolites. Both upregulation and downregulation of dLdh in neurons resulted in decreased survival and memory impairment with age. Glial downregulation of dLdh expression caused age-related memory impairment without altering survival, while upregulated glial dLdh expression lowered survival without disrupting memory. Both neuronal and glial dLdh upregulation increased neutral lipid accumulation. We provide evidence that altered lactate metabolism with age affects the tricarboxylic acid (TCA) cycle, 2-hydroxyglutarate (2HG), and neutral lipid accumulation. Collectively, our findings indicate that the direct alteration of lactate metabolism in either glia or neurons affects memory and survival but only in an age-dependent manner.
Topics: Animals; L-Lactate Dehydrogenase; Drosophila melanogaster; Neuroglia; Neurons; Astrocytes; Memory Disorders; Lactic Acid; Lipids
PubMed: 36849157
DOI: 10.18632/aging.204565 -
Brain Pathology (Zurich, Switzerland) Jan 2016There are over 120 types of brain tumor and approximately 45% of primary brain tumors are gliomas, of which glioblastoma multiforme (GBM) is the most common and... (Review)
Review
There are over 120 types of brain tumor and approximately 45% of primary brain tumors are gliomas, of which glioblastoma multiforme (GBM) is the most common and aggressive with a median survival rate of 14 months. Despite progress in our knowledge, current therapies are unable to effectively combat primary brain tumors and patient survival remains poor. Tumor metabolism is important to consider in therapeutic approaches and is the focus of numerous research investigations. Lactate dehydrogenase A (LDHA) is a cytosolic enzyme, predominantly involved in anaerobic and aerobic glycolysis (the Warburg effect); however, it has multiple additional functions in non-neoplastic and neoplastic tissues, which are not commonly known or discussed. This review summarizes what is currently known about the function of LDHA and identifies areas that would benefit from further exploration. The current knowledge of the role of LDHA in the brain and its potential as a therapeutic target for brain tumors will also be highlighted. The Warburg effect appears to be universal in tumors, including primary brain tumors, and LDHA (because of its involvement with this process) has been identified as a potential therapeutic target. Currently, there are, however, no suitable LDHA inhibitors available for tumor therapies in the clinic.
Topics: Animals; Brain Neoplasms; Humans; Isoenzymes; L-Lactate Dehydrogenase; Lactate Dehydrogenase 5
PubMed: 26269128
DOI: 10.1111/bpa.12299