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Science (New York, N.Y.) Mar 2023Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low...
Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions. Analysis of 33 enzymes from human carbohydrate metabolism identified 830 protein-metabolite interactions, including known regulators, substrates, and products as well as previously unreported interactions. We functionally validated a subset of interactions, including the isoform-specific inhibition of lactate dehydrogenase by long-chain acyl-coenzyme A. Cell treatment with fatty acids caused a loss of pyruvate-lactate interconversion dependent on lactate dehydrogenase isoform expression. These protein-metabolite interactions may contribute to the dynamic, tissue-specific metabolic flexibility that enables growth and survival in an ever-changing nutrient environment.
Topics: Humans; Carbohydrate Metabolism; Fatty Acids; L-Lactate Dehydrogenase; Organ Specificity; Metabolome; Mass Spectrometry; Allosteric Regulation
PubMed: 36893255
DOI: 10.1126/science.abm3452 -
Cell Jun 2019RLR-mediated type I IFN production plays a pivotal role in elevating host immunity for viral clearance and cancer immune surveillance. Here, we report that glycolysis,...
RLR-mediated type I IFN production plays a pivotal role in elevating host immunity for viral clearance and cancer immune surveillance. Here, we report that glycolysis, which is inactivated during RLR activation, serves as a barrier to impede type I IFN production upon RLR activation. RLR-triggered MAVS-RIG-I recognition hijacks hexokinase binding to MAVS, leading to the impairment of hexokinase mitochondria localization and activation. Lactate serves as a key metabolite responsible for glycolysis-mediated RLR signaling inhibition by directly binding to MAVS transmembrane (TM) domain and preventing MAVS aggregation. Notably, lactate restoration reverses increased IFN production caused by lactate deficiency. Using pharmacological and genetic approaches, we show that lactate reduction by lactate dehydrogenase A (LDHA) inactivation heightens type I IFN production to protect mice from viral infection. Our study establishes a critical role of glycolysis-derived lactate in limiting RLR signaling and identifies MAVS as a direct sensor of lactate, which functions to connect energy metabolism and innate immunity.
Topics: Adaptor Proteins, Signal Transducing; Animals; DEAD Box Protein 58; Female; Glycolysis; HEK293 Cells; Humans; Interferon-beta; L-Lactate Dehydrogenase; Lactic Acid; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; RAW 264.7 Cells; Receptors, Cell Surface; Receptors, Immunologic; Signal Transduction; Transfection
PubMed: 31155231
DOI: 10.1016/j.cell.2019.05.003 -
Science (New York, N.Y.) Sep 2022Gain-of-function mutations in isocitrate dehydrogenase (IDH) in human cancers result in the production of d-2-hydroxyglutarate (d-2HG), an oncometabolite that promotes...
Gain-of-function mutations in isocitrate dehydrogenase (IDH) in human cancers result in the production of d-2-hydroxyglutarate (d-2HG), an oncometabolite that promotes tumorigenesis through epigenetic alterations. The cancer cell-intrinsic effects of d-2HG are well understood, but its tumor cell-nonautonomous roles remain poorly explored. We compared the oncometabolite d-2HG with its enantiomer, l-2HG, and found that tumor-derived d-2HG was taken up by CD8 T cells and altered their metabolism and antitumor functions in an acute and reversible fashion. We identified the glycolytic enzyme lactate dehydrogenase (LDH) as a molecular target of d-2HG. d-2HG and inhibition of LDH drive a metabolic program and immune CD8 T cell signature marked by decreased cytotoxicity and impaired interferon-γ signaling that was recapitulated in clinical samples from human patients with mutant gliomas.
Topics: Animals; CD8-Positive T-Lymphocytes; Carcinogenesis; Gain of Function Mutation; Glutarates; Humans; Interferon-gamma; Isocitrate Dehydrogenase; L-Lactate Dehydrogenase; Mice; Neoplasms
PubMed: 36173860
DOI: 10.1126/science.abj5104 -
Nature Communications Feb 2023Alterations in energy metabolism are associated with depression. However, the role of glycolysis in the pathogenesis of depression and the underlying molecular...
Alterations in energy metabolism are associated with depression. However, the role of glycolysis in the pathogenesis of depression and the underlying molecular mechanisms remain unexplored. Through an unbiased proteomic screen coupled with biochemical verifications, we show that the levels of glycolysis and lactate dehydrogenase A (LDHA), a glycolytic enzyme that catalyzes L-lactate production, are reduced in the dorsomedial prefrontal cortex (dmPFC) of stress-susceptible mice in chronic social defeat stress (CSDS) model. Conditional knockout of LDHA from the brain promotes depressive-like behaviors in both male and female mice, accompanied with reduced L-lactate levels and decreased neuronal excitability in the dmPFC. Moreover, these phenotypes could be duplicated by knockdown of LDHA in the dmPFC or specifically in astrocytes. In contrast, overexpression of LDHA reverses these phenotypic changes in CSDS-susceptible mice. Mechanistic studies demonstrate that L-lactate promotes neuronal excitability through monocarboxylic acid transporter 2 (MCT2) and by inhibiting large-conductance Ca-activated potassium (BK) channel. Together, these results reveal a role of LDHA in maintaining neuronal excitability to prevent depressive-like behaviors.
Topics: Mice; Male; Female; Animals; Lactate Dehydrogenase 5; Astrocytes; Lactic Acid; L-Lactate Dehydrogenase; Proteomics; Carrier Proteins
PubMed: 36759610
DOI: 10.1038/s41467-023-36209-5 -
Nature Communications Sep 2022Adipose tissue macrophage (ATM) inflammation is involved with meta-inflammation and pathology of metabolic complications. Here we report that in adipocytes, elevated...
Adipose tissue macrophage (ATM) inflammation is involved with meta-inflammation and pathology of metabolic complications. Here we report that in adipocytes, elevated lactate production, previously regarded as the waste product of glycolysis, serves as a danger signal to promote ATM polarization to an inflammatory state in the context of obesity. Adipocyte-selective deletion of lactate dehydrogenase A (Ldha), the enzyme converting pyruvate to lactate, protects mice from obesity-associated glucose intolerance and insulin resistance, accompanied by a lower percentage of inflammatory ATM and reduced production of pro-inflammatory cytokines such as interleukin 1β (IL-1β). Mechanistically, lactate, at its physiological concentration, fosters the activation of inflammatory macrophages by directly binding to the catalytic domain of prolyl hydroxylase domain-containing 2 (PHD2) in a competitive manner with α-ketoglutarate and stabilizes hypoxia inducible factor (HIF-1α). Lactate-induced IL-1β was abolished in PHD2-deficient macrophages. Human adipose lactate level is positively linked with local inflammatory features and insulin resistance index independent of the body mass index (BMI). Our study shows a critical function of adipocyte-derived lactate in promoting the pro-inflammatory microenvironment in adipose and identifies PHD2 as a direct sensor of lactate, which functions to connect chronic inflammation and energy metabolism.
Topics: Adipocytes; Adipose Tissue; Animals; Humans; Hypoxia-Inducible Factor-Proline Dioxygenases; Inflammation; Insulin Resistance; L-Lactate Dehydrogenase; Lactate Dehydrogenase 5; Lactic Acid; Macrophages; Mice; Obesity; Procollagen-Proline Dioxygenase; Prolyl Hydroxylases
PubMed: 36064857
DOI: 10.1038/s41467-022-32871-3 -
Proceedings of the National Academy of... Mar 2020Interleukin (IL)-2 and IL-21 dichotomously shape CD8 T cell differentiation. IL-2 drives terminal differentiation, generating cells that are poorly effective against...
Interleukin (IL)-2 and IL-21 dichotomously shape CD8 T cell differentiation. IL-2 drives terminal differentiation, generating cells that are poorly effective against tumors, whereas IL-21 promotes stem cell memory T cells (T) and antitumor responses. Here we investigated the role of metabolic programming in the developmental differences induced by these cytokines. IL-2 promoted effector-like metabolism and aerobic glycolysis, robustly inducing lactate dehydrogenase (LDH) and lactate production, whereas IL-21 maintained a metabolically quiescent state dependent on oxidative phosphorylation. LDH inhibition rewired IL-2-induced effects, promoting pyruvate entry into the tricarboxylic acid cycle and inhibiting terminal effector and exhaustion programs, including mRNA expression of members of the NR4A family of nuclear receptors, as well as and While deletion of prevented development of cells with antitumor effector function, transient LDH inhibition enhanced the generation of memory cells capable of triggering robust antitumor responses after adoptive transfer. LDH inhibition did not significantly affect IL-21-induced metabolism but caused major transcriptomic changes, including the suppression of IL-21-induced exhaustion markers LAG3, PD1, 2B4, and TIM3. LDH inhibition combined with IL-21 increased the formation of T cells, resulting in more profound antitumor responses and prolonged host survival. These findings indicate a pivotal role for LDH in modulating cytokine-mediated T cell differentiation and underscore the therapeutic potential of transiently inhibiting LDH during adoptive T cell-based immunotherapy, with an unanticipated cooperative antitumor effect of LDH inhibition and IL-21.
Topics: Animals; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Line, Tumor; Enzyme Inhibitors; Humans; Immunologic Memory; Immunotherapy, Adoptive; Interleukin-2; Interleukins; L-Lactate Dehydrogenase; Melanoma, Experimental; Mice; Primary Cell Culture; Stem Cells
PubMed: 32123114
DOI: 10.1073/pnas.1920413117 -
Theranostics 2022Chemoresistance to 5-fluorouracil (5-FU) is a major barrier to influence the treatment efficiency of colorectal cancer (CRC) patients, while the precise molecular...
Chemoresistance to 5-fluorouracil (5-FU) is a major barrier to influence the treatment efficiency of colorectal cancer (CRC) patients, while the precise molecular mechanisms underlying 5-FU resistance remain to be fully elucidated. The metabolic profiles including ATP generation, glucose consumption, lactate generation, and oxygen consumption rate (OCR) in 5-FU resistant CRC cells were compared with those in their parental cells. Subsequently, a series of and experiments were carried out to investigate the mechanisms responsible for metabolic reprogramming of 5-FU resistant CRC cells. We found that 5-FU resistant CRC cells showed increased levels of ATP generation, glucose consumption, lactate generation, and OCR as compared with those in their parental cells. Further, increased levels of mRNA N-methyladenosine (mA) and methyltransferase-like 3 (METTL3) were observed in 5-FU resistant CRC cells. Inhibition or knockdown of METTL3 can suppress glycolysis and restore chemosensitivity of 5-FU resistant CRC cells. Mechanistically, METTL3 enhances the expression of LDHA, which catalyzes the conversion of pyruvate to lactate, to trigger glycolysis and 5-FU resistance. METTL3 can increase the transcription of LDHA via stabilizing mRNA of hypoxia-inducible factor (HIF-1α), further, METTL3 also triggers the translation of LDHA mRNA via methylation of its CDS region and recruitment of YTH domain-containing family protein 1 (YTHDF1). Targeted inhibition of METTL3/LDHA axis can significantly increase the and 5-FU sensitivity of CRC cells. Our study indicates that METTL3/LDHA axis-induced glucose metabolism is a potential therapy target to overcome 5-FU resistance in CRC cells.
Topics: Adenosine; Adenosine Triphosphate; Cell Line, Tumor; Colorectal Neoplasms; Drug Resistance, Neoplasm; Fluorouracil; Glucose; HCT116 Cells; Humans; L-Lactate Dehydrogenase; Lactates; Methyltransferases; RNA, Messenger
PubMed: 35832094
DOI: 10.7150/thno.73746 -
Blood May 2022Translation is essential for megakaryocyte (MK) maturation and platelet production. However, how the translational pathways are regulated in this process remains...
Translation is essential for megakaryocyte (MK) maturation and platelet production. However, how the translational pathways are regulated in this process remains unknown. In this study, we found that MK/platelet-specific lactate dehydrogenase A (LdhA) knockout mice exhibited an increased number of platelets with remarkably accelerated MK maturation and proplatelet formation. Interestingly, the role of LDHA in MK maturation and platelet formation did not depend on lactate content, which was the major product of LDHA. Mechanism studies revealed that LDHA interacted with eukaryotic elongation factor 2 (eEF2) in the cytoplasm, controlling the participation of eEF2 in translation at the ribosome. Furthermore, the interaction of LDHA and eEF2 was dependent on nicotinamide adenine dinucleotide (NADH), a coenzyme of LDHA. NADH-competitive inhibitors of LDHA could release eEF2 from the LDHA pool, upregulate translation, and enhance MK maturation in vitro. Among LDHA inhibitors, stiripentol significantly promoted the production of platelets in vivo under a physiological state and in the immune thrombocytopenia model. Moreover, stiripentol could promote platelet production from human cord blood mononuclear cell-derived MKs and also have a superposed effect with romiplostim. In short, this study shows a novel nonclassical function of LDHA in translation that may serve as a potential target for thrombocytopenia therapy.
Topics: Animals; Blood Platelets; Elongation Factor 2 Kinase; Enzyme Inhibitors; L-Lactate Dehydrogenase; Megakaryocytes; Mice; Mice, Knockout; NAD; Peptide Elongation Factor 2; Thrombocytopenia; Thrombopoiesis
PubMed: 35176139
DOI: 10.1182/blood.2022015620 -
Nature Metabolism Dec 2022The glycolytic enzyme lactate dehydrogenase A (LDHA) is frequently overexpressed in cancer, which promotes glycolysis and cancer. The oncogenic effect of LDHA has been...
The glycolytic enzyme lactate dehydrogenase A (LDHA) is frequently overexpressed in cancer, which promotes glycolysis and cancer. The oncogenic effect of LDHA has been attributed to its glycolytic enzyme activity. Here we report an unexpected noncanonical oncogenic mechanism of LDHA; LDHA activates small GTPase Rac1 to promote cancer independently of its glycolytic enzyme activity. Mechanistically, LDHA interacts with the active form of Rac1, Rac1-GTP, to inhibit Rac1-GTP interaction with its negative regulator, GTPase-activating proteins, leading to Rac1 activation in cancer cells and mouse tissues. In clinical breast cancer specimens, LDHA overexpression is associated with higher Rac1 activity. Rac1 inhibition suppresses the oncogenic effect of LDHA. Combination inhibition of LDHA enzyme activity and Rac1 activity by small-molecule inhibitors displays a synergistic inhibitory effect on breast cancers with LDHA overexpression. These results reveal a critical oncogenic mechanism of LDHA and suggest a promising therapeutic strategy for breast cancers with LDHA overexpression.
Topics: Animals; Mice; Lactate Dehydrogenase 5; L-Lactate Dehydrogenase; GTP Phosphohydrolases; Isoenzymes; Neoplasms; Guanosine Triphosphate
PubMed: 36536137
DOI: 10.1038/s42255-022-00708-4 -
Journal of Experimental & Clinical... Aug 2020Lysine succinylation is an emerging posttranslational modification that has garnered increased attention recently, but its role in gastric cancer (GC) remains...
BACKGROUND
Lysine succinylation is an emerging posttranslational modification that has garnered increased attention recently, but its role in gastric cancer (GC) remains underexplored.
METHODS
Proteomic quantification of lysine succinylation was performed in human GC tissues and adjacent normal tissues by mass spectrometry. The mRNA and protein levels of lactate dehydrogenase A (LDHA) in GC and adjacent normal tissues were analyzed by qRT-PCR and western blot, respectively. The expression of K222-succinylated LDHA was measured in GC tissue microarray by the K222 succinylation-specific antibody. The interaction between LDHA and sequestosome 1 (SQSTM1) was measured by co-immunoprecipitation (co-IP) and proximity ligation assay (PLA). The binding of carnitine palmitoyltransferase 1A (CPT1A) to LDHA was determined by co-IP. The effect of K222-succinylated LDHA on tumor growth and metastasis was evaluated by in vitro and in vivo experiments.
RESULTS
Altogether, 503 lysine succinylation sites in 303 proteins were identified. Lactate dehydrogenase A (LDHA), the key enzyme in Warburg effect, was found highly succinylated at K222 in GC. Intriguingly, this modification did not affect LDHA ubiquitination, but reduced the binding of ubiquitinated LDHA to SQSTM1, thereby decreasing its lysosomal degradation. We demonstrated that CPT1A functions as a lysine succinyltransferase that interacts with and succinylates LDHA. Moreover, high K222-succinylation of LDHA was associated with poor prognosis in patients with GC. Finally, overexpression of a succinylation-mimic mutant of LDHA promoted cell proliferation, invasion, and migration.
CONCLUSIONS
Our data revealed a novel lysosomal pathway of LDHA degradation, which is mediated by the binding of K63-ubiquitinated LDHA to SQSTM1. Strikingly, CPT1A succinylates LDHA on K222, which thereby reduces the binding and inhibits the degradation of LDHA, as well as promotes GC invasion and proliferation. This study thus uncovers a new role of lysine succinylation and the mechanism underlying LDHA upregulation in GC.
Topics: Animals; Apoptosis; Biomarkers, Tumor; Carnitine O-Palmitoyltransferase; Cell Proliferation; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; L-Lactate Dehydrogenase; Lung Neoplasms; Lysine; Lysosomes; Male; Melanoma; Mice; Mice, Nude; Middle Aged; Prognosis; Protein Processing, Post-Translational; Proteolysis; Sequestosome-1 Protein; Stomach Neoplasms; Succinic Acid; Survival Rate; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
PubMed: 32859246
DOI: 10.1186/s13046-020-01681-0