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Oxidation Resistance 1 Modulates Glycolytic Pathways in the Cerebellum via an Interaction with Glucose-6-Phosphate Isomerase.

ABSTRACT: Glucose metabolism is essential for the brain: it not only provides the required energy for cellular function and communication but also participates in balancing the levels of oxidative stress in neurons. Defects in glucose metabolism have been described in neurodegenerative disease; however, it remains unclear how this fundamental process contributes to neuronal cell death in these disorders. Here, we investigated the molecular mechanisms driving the selective neurodegeneration in an ataxic mouse model lacking oxidation resistance 1 (Oxr1) and discovered an unexpected function for this protein as a regulator of the glycolytic enzyme, glucose-6-phosphate isomerase (GPI/Gpi1). Initially, we present a dysregulation of metabolites of glucose metabolism at the pre-symptomatic stage in the Oxr1 knockout cerebellum. We then demonstrate that Oxr1 and Gpi1 physically and functionally interact and that the level of Gpi1 oligomerisation is disrupted when Oxr1 is deleted in vivo. Furthermore, we show that Oxr1 modulates the additional and less well-understood roles of Gpi1 as a cytokine and neuroprotective factor. Overall, our data identify a new molecular function for Oxr1, establishing this protein as important player in neuronal survival, regulating both oxidative stress and glucose metabolism in the brain.

SUBMITTER: Finelli MJ

PROVIDER: S-EPMC6368252 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND/AIM:Nearly all mammalian tumors of diverse tissues are believed to be dependent on fermentative glycolysis, marked by elevated production of lactic acid and expression of glycolytic enzymes, most notably lactic acid dehydrogenase (LDH). Therefore, there has been significant interest in developing chemotherapy drugs that selectively target various isoforms of the LDH enzyme. However, considerable questions remain as to the consequences of biological ablation of LDH or upstream targeting of the glycolytic pathway. MATERIALS AND METHODS:In this study, we explore the biochemical and whole transcriptomic effects of CRISPR-Cas9 gene knockout (KO) of lactate dehydrogenases A and B [LDHA/B double KO (DKO)] and glucose-6-phosphate isomerase (GPI KO) in the human colon cancer cell line LS174T, using Affymetrix 2.1 ST arrays. RESULTS:The metabolic biochemical profiles corroborate that relative to wild type (WT), LDHA/B DKO produced no lactic acid, (GPI KO) produced minimal lactic acid and both KOs displayed higher mitochondrial respiration, and minimal use of glucose with no loss of cell viability. These findings show a high biochemical energy efficiency as measured by ATP in glycolysis-null cells. Next, transcriptomic analysis conducted on 48,226 mRNA transcripts reflect 273 differentially expressed genes (DEGS) in the GPI KO clone set, 193 DEGS in the LDHA/B DKO clone set with 47 DEGs common to both KO clones. Glycolytic-null cells reflect up-regulation in gene transcripts typically associated with nutrient deprivation / fasting and possible use of fats for energy: thioredoxin interacting protein (TXNIP), mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), PPAR? coactivator 1? (PGC-1?), and acetyl-CoA acyltransferase 2 (ACAA2). Other changes in non-ergometric transcripts in both KOs show losses in "stemness", WNT signaling pathway, chemo/radiation resistance, retinoic acid synthesis, drug detoxification, androgen/estrogen activation, and extracellular matrix reprogramming genes. CONCLUSION:These findings demonstrate that: 1) The "Warburg effect" is dispensable, 2) loss of the LDHAB gene is not only inconsequential to viability but fosters greater mitochondrial energy, and 3) drugs that target LDHA/B are likely to be ineffective without a plausible combination second drug target.

Project description:For a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key regulatory enzyme of this pathway and can produce ATP through oxidative level of phosphorylation. Previously, we reported that GAPDH purified from a variety of malignant tissues, but not from normal tissues, was strongly inactivated by a normal metabolite, methylglyoxal (MG). Molecular mechanism behind MG mediated GAPDH inhibition in cancer cells is not well understood.GAPDH was purified from Ehrlich ascites carcinoma (EAC) cells based on its enzymatic activity. GAPDH associated proteins in EAC cells and 3-methylcholanthrene (3MC) induced mouse tumor tissue were detected by mass spectrometry analysis and immunoprecipitation (IP) experiment, respectively. Interacting domains of GAPDH and its associated proteins were assessed by in silico molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in cancer cells was evaluated by measuring enzyme activity, Circular dichroism (CD) spectroscopy, IP and mass spectrometry analyses.Here, we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However, both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 mM MG, association of GAPDH with PKM2 or GPI is not perturbed, but the enzymatic activity of GAPDH is reduced to 26.8 ± 5 % in 3MC induced tumor and 57.8 ± 2.3 % in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only, and changes the secondary structure of the protein complex.PKM2 may regulate the enzymatic activity of GAPDH. Increased enzymatic activity of GAPDH in tumor cells may be attributed to its association with PKM2 and GPI. Association of GAPDH with PKM2 and GPI could be a signature for cancer cells. Glycation at R399 of PKM2 and changes in the secondary structure of GAPDH complex could be one of the mechanisms by which GAPDH activity is inhibited in tumor cells by MG.

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Oxidation Resistance 1 Modulates Glycolytic Pathways in the Cerebellum via an Interaction with Glucose-6-Phosphate Isomerase.

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