Sod1 integrates oxygen availability to redox regulate NADPH production and the thiol redoxome

Cu/Zn superoxide dismutase (Sod1) is a highly conserved and abundant antioxidant enzyme that detoxifies superoxide (O2•−) by catalyzing its conversion to dioxygen (O2) and hydrogen peroxide (H2O2). Using Saccharomyces cerevisiae and mammalian cells, we discovered that a major aspect of the antioxidant function of Sod1 is to integrate O2 availability to promote NADPH production. The mechanism involves Sod1-derived H2O2 oxidatively inactivating the glycolytic enzyme, GAPDH, which in turn reroutes carbohydrate flux to the oxidative phase of the pentose phosphate pathway (oxPPP) to generate NADPH. The aerobic oxidation of GAPDH is dependent on and rate-limited by Sod1. Thus, Sod1 senses O2 via O2•− to balance glycolytic and oxPPP flux, through control of GAPDH activity, for adaptation to life in air. Importantly, this mechanism for Sod1 antioxidant activity requires the bulk of cellular Sod1, unlike for its role in protection against O2•− toxicity, which only requires <1% of total Sod1. Using mass spectrometry, we identified proteome-wide targets of Sod1-dependent redox signaling, including numerous metabolic enzymes. Altogether, Sod1-derived H2O2 is important for antioxidant defense and a master regulator of metabolism and the thiol redoxome.

Growth of Mycobacterium tuberculosis at acidic pH depends on lipid assimilation and is accompanied by reduced GAPDH activity

Acidic pH arrests the growth of Mycobacterium tuberculosis in vitro (pH < 5.8) and is thought to significantly contribute to the ability of macrophages to control M. tuberculosis replication. However, this pathogen has been shown to survive and even slowly replicate within macrophage phagolysosomes (pH 4.5 to 5) [M. S. Gomes et al., Infect. Immun. 67, 3199–3206 (1999)] [S. Levitte et al., Cell Host Microbe 20, 250–258 (2016)]. Here, we demonstrate that M. tuberculosis can grow at acidic pH, as low as pH 4.5, in the presence of host-relevant lipids. We show that lack of phosphoenolpyruvate carboxykinase and isocitrate lyase, two enzymes necessary for lipid assimilation, is cidal to M. tuberculosis in the presence of oleic acid at acidic pH. Metabolomic analysis revealed that M. tuberculosis responds to acidic pH by altering its metabolism to preferentially assimilate lipids such as oleic acid over carbohydrates such as glycerol. We show that the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is impaired in acid-exposed M. tuberculosis likely contributing to a reduction in glycolytic flux. The generation of endogenous reactive oxygen species at acidic pH is consistent with the inhibition of GAPDH, an enzyme well-known to be sensitive to oxidation. This work shows that M. tuberculosis alters its carbon diet in response to pH and provides a greater understanding of the physiology of this pathogen during acid stress.

The Combined Influence of Magnesium and Insulin on Central Metabolic Functions and Expression of Genes Involved in Magnesium Homeostasis of Cultured Bovine Adipocytes

At the onset of lactation, dairy cows suffer from insulin resistance, insulin deficiency or both, similar to human diabetes, resulting in lipolysis, ketosis and fatty liver. This work explored the combined effects of different levels of magnesium (0.1, 0.3, 1 and 3 mM) and insulin (25, 250 and 25,000 pM) on metabolic pathways and the expression of magnesium-responsive genes in a bovine adipocyte model. Magnesium starvation (0.1 mM) and low insulin (25 pM) independently decreased or tended to decrease the accumulation of non-polar lipids and uptake of the glucose analog 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-deoxyglucose (6-NBDG). Activity of glycerol 3-phosphate dehydrogenase (GPDH) was highest at 25 pM insulin and 3 mM magnesium. Expression of SLC41A1 and SLC41A3 was reduced at 0.1 mM magnesium either across insulin concentrations (SLC41A1) or at 250 pM insulin (SLC41A3). MAGT1 expression was reduced at 3 mM magnesium. NIPA1 expression was reduced at 3 mM and 0.1 mM magnesium at 25 and 250 pM insulin, respectively. Expression of SLC41A2, CNNM2, TRPM6 and TRPM7 was not affected. We conclude that magnesium promotes lipogenesis in adipocytes and inversely regulates the transcription of genes that increase vs. decrease cytosolic magnesium concentration. The induction of GAPDH activity by surplus magnesium at low insulin concentration can counteract excessive lipomobilization.

Enzymatic Analysis of Yeast Cell Wall-Resident GAPDH and Its Secretion

ABSTRACT In yeast, many proteins are found in both the cytoplasmic and extracellular compartments, and consequently it can be difficult to distinguish nonconventional secretion from cellular leakage. Therefore, we monitored the extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity of intact cells as a specific marker for nonconventional secretion. Extracellular GAPDH activity was proportional to the number of cells assayed, increased with incubation time, and was dependent on added substrates. Preincubation of intact cells with 100 μM dithiothreitol increased the reaction rate, consistent with increased access of the enzyme after reduction of cell wall disulfide cross-links. Such treatment did not increase cell permeability to propidium iodide, in contrast to effects of higher concentrations of reducing agents. An amine-specific membrane-impermeant biotinylation reagent specifically inactivated extracellular GAPDH. The enzyme was secreted again after a 30- to 60-min lag following the inactivation, and there was no concomitant increase in propidium iodide staining. There were about 4 × 104 active GAPDH molecules per cell at steady state, and secretion studies showed replenishment to that level 1 h after inactivation. These results establish conditions for specific quantitative assays of cell wall proteins in the absence of cytoplasmic leakage and for subsequent quantification of secretion rates in intact cells. IMPORTANCE Eukaryotic cells secrete many proteins, including many proteins that do not follow the classical secretion pathway. Among these, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is unexpectedly found in the walls of yeasts and other fungi and in extracellular space in mammalian cell cultures. It is difficult to quantify extracellular GAPDH, because leakage of just a little of the very large amount of cytoplasmic enzyme can invalidate the determinations. We used enzymatic assays of intact cells while also maintaining membrane integrity. The results lead to estimates of the amount of extracellular enzyme and its rate of secretion to the wall in intact cells. Therefore, enzyme assays under controlled conditions can be used to investigate nonconventional secretion more generally.

Overproduction of Chloroplast Glyceraldehyde-3-Phosphate Dehydrogenase Improves Photosynthesis Slightly under Elevated [CO2] Conditions in Rice

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) limits the regeneration of ribulose 1,5-bisphosphate (RuBP) in the Calvin–Benson cycle. However, it does not always limit the rate of CO2 assimilation. In the present study, the effects of overproduction of GAPDH on the rate of CO2 assimilation under elevated [CO2] conditions, where the capacity for RuBP regeneration limits photosynthesis, were examined in transgenic rice (Oryza sativa). GAPDH activity was increased to 3.2- and 4.5-fold of the wild-type levels by co-overexpression of the GAPDH genes, GAPA and GAPB, respectively. In the transgenic rice plants, the rate of CO2 assimilation under elevated [CO2] conditions increased by approximately 10%, whereas that under normal and low [CO2] conditions was not affected. These results indicate that overproduction of GAPDH is effective in improving photosynthesis under elevated [CO2] conditions, although its magnitude is relatively small. By contrast, biomass production of the transgenic rice plants was not greater than that of wild-type plants under elevated [CO2] conditions, although starch content tended to increase marginally.

Abstract 14312: Hexokinase 1 Cellular Localization Regulates the Metabolic Fate of Glucose

Introduction: The product of hexokinase (HK) enzymes, glucose-6-phosphate (G6P), can be metabolized through glycolysis or directed to alternative pathways, such as the pentose-phosphate-pathway (PPP) for anabolism. However, it is not known what determines the fate of G6P. HK1 contains an N-terminal mitochondrial-binding domain, but its physiologic significance remains unclear. Inflammation is a tightly controlled process sensitive to dynamic changes in the tissue environment and the intrinsic state of immune cells, both contributing to the initiation and resolution of inflammation. The loss of HK1 attenuates glycolytic reprogramming and inflammatory cytokine production in LPS stimulated macrophages. Given the importance of HK1 in the innate immune response, we used myeloid cells as a model system to study the effect of HK1 subcellular localization on cellular metabolism and inflammation. Results: We overexpressed full-length and truncated HK1 in tissue culture and generated mice lacking the HK1 mitochondrial-binding domain (ΔE1HK1). Although ΔE1HK1 mice displayed no overt phenotype, HK1 dislocation from the mitochondria increased glucose flux through the PPP, decreased flux below the level of GAPDH, and induced a hyper-inflammatory response to lipopolysaccharide. The mechanism for the increased PPP flux is through a glycolytic block at GAPDH, which is mediated by binding of cytosolic HK1 with S100A8/A9 and increased GAPDH nitrosylation through iNOS. Human and mouse macrophages from conditions of low-grade inflammation, such as aging and diabetes, displayed an increase in cytosolic HK1 and cytokine production, along with reduced GAPDH activity. Conclusions: Our data indicate that HK1 subcellular localization is a critical regulator of glucose metabolism and determines whether glucose is shuttled into PPP at the expense of glycolysis, and regulates the inflammatory response in macrophages (Figure).

Enzymatic analysis of yeast cell wall-resident GAPDH and its secretion

In yeast, many proteins are found both in the cytoplasmic and extracellular compartments, and consequently it can be difficult to distinguish non-conventional secretion from cellular leakage. We therefore monitored extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity of intact cells as a specific marker for non-conventional secretion. Extracellular GAPDH activity was proportional to the number of cells assayed, increased with incubation time, and was dependent on added substrates. Preincubation of intact cells with 100μM dithiothreitol increased the reaction rate, consistent with increased access of the enzyme after reduction of cell wall disulfide crosslinks. Such treatment did not increase cell permeability to propidium iodide, in contrast to effects of higher concentrations of reducing agents. An amine-specific membrane-impermeant biotinylation reagent specifically inactivated extracellular GAPDH. The enzyme was secreted again after a 30-60-minute lag following the inactivation, and there was no concomitant increase in propidium iodide staining. There were about 4 × 104 active GAPDH molecules per cell at steady state, and secretion studies showed replenishment to that level one hour after inactivation. These results establish conditions for specific quantitative assays of cell wall proteins in the absence of cytoplasmic leakage and for subsequent quantification of secretion rates in intact cells.ImportanceEukaryotic cells secrete many proteins, including many proteins that do not follow the classical secretion pathway. Among these, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is unexpectedly found in the walls of yeasts and other fungi, and in extracellular space in mammalian cell cultures. It is difficult to quantify extracellular GAPDH, because leakage of just a little of the very large amount of cytoplasmic enzyme can invalidate the determinations. We used enzymatic assays of intact cells, while also maintaining membrane integrity. The results lead to estimates of the amount of extracellular enzyme, and its rate of secretion to the wall in intact cells. Therefore, enzyme assays under controlled conditions can be used to investigate non-conventional secretion more generally.

Deciphering the Binding of Salicylic Acid to Arabidopsis thaliana Chloroplastic GAPDH-A1

Salicylic acid (SA) has an essential role in the responses of plants to pathogens. SA initiates defence signalling via binding to proteins. NPR1 is a transcriptional co-activator and a key target of SA binding. Many other proteins have recently been shown to bind SA. Amongst these proteins are important enzymes of primary metabolism. This fact could stand behind SA’s ability to control energy fluxes in stressed plants. Nevertheless, only sparse information exists on the role and mechanisms of such binding. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was previously demonstrated to bind SA both in human and plants. Here, we detail that the A1 isomer of chloroplastic glyceraldehyde 3-phosphate dehydrogenase (GAPA1) from Arabidopsis thaliana binds SA with a KD of 16.7 nM, as shown in surface plasmon resonance experiments. Besides, we show that SA inhibits its GAPDH activity in vitro. To gain some insight into the underlying molecular interactions and binding mechanism, we combined in silico molecular docking experiments and molecular dynamics simulations on the free protein and protein–ligand complex. The molecular docking analysis yielded to the identification of two putative binding pockets for SA. A simulation in water of the complex between SA and the protein allowed us to determine that only one pocket—a surface cavity around Asn35—would efficiently bind SA in the presence of solvent. In silico mutagenesis and simulations of the ligand/protein complexes pointed to the importance of Asn35 and Arg81 in the binding of SA to GAPA1. The importance of this is further supported through experimental biochemical assays. Indeed, mutating GAPA1 Asn35 into Gly or Arg81 into Leu strongly diminished the ability of the enzyme to bind SA. The very same cavity is responsible for the NADP+ binding to GAPA1. More precisely, modelling suggests that SA binds to the very site where the pyrimidine group of the cofactor fits. NADH inhibited in a dose-response manner the binding of SA to GAPA1, validating our data.

614. Metabolic Interventions for the Resensitization of Daptomycin-Resistant (DAP-R) Streptococcus mitis-oralis Strains to DAP In vitro and Ex Vivo in a Simulated Model of Experimental Endocarditis (IE)

Background Streptococcus mitis-oralis is a leading cause of IE. Treatment of this pathogen is limited by frequent high-level β-lactam resistance and the propensity to develop high-level DAP-R during DAP exposure. The current study elucidated key metabolic perturbations associated with high-level DAP-R in prototype S. mitis-oralis strain 351, following in vitro selection of DAP-R by 10-day serial passage in sub-inhibitory DAP. Furthermore, to test translatability of such metabolic changes (see below), the synergistic activity of combinations of DAP plus a strategic metabolic inhibitor (i.e., fosfomycin) vs. DAP or fosfomycin alone was assessed, using DAP-R S. mitis-oralis strain 351-D10 (MIC >256 µg/mL) in vitro and in an ex vivo IE model. Methods MICs. E test Growth Curve: Optical density (OD600) determined spectrophotometrically at 0–8 hours glyceraldehyde-3-phosphatedehydrogenase (GAPDH) activity. Kit from BioVision®. Metabolomics: one-dimensional 1H NMR-MS and two-dimensional 1H-13C HSQC in vitro time-kill assay: Using sub-MIC/MIC drug concentrations (initial inoculum ~1 × 105 CFU/mL) for 0, 2, 4, 6, and 8 hours. Ex vivo IE model: Simulated endocardial vegetations (SEVs) quantitatively cultured at 0, 4, 8, 24, 32, 48, and 72 hours with DAP or fosfomycin alone or in combination. Results NMR metabolomics analysis identified a number of metabolite differences in the 351 D10 DAP-R vs. 351 DAP-S strain (Figure 1). These data are consistent with a significant reduction in GAPDH activity (a glycolytic enzyme) in 351-D10 vs. 351 strain. Based on these metabolic changes, fosfomycin (a phosphoenolpyruvate analog) was chosen as a strategic metabolic inhibitor to attempt to “resensitize” our DAP-R S. mitis-oralis strain to DAP. The combination of DAP + fosfomycin demonstrated synergistic killing of the DAP-R strain vs. DAP or fosfomycin alone in the in vitro time-killing assays. Moreover, the DAP-R strain was synergistically cleared from SEVs by DAP + fosfomycin in the ex vivo IE model. Conclusion Taken together, these data indicate there are unique metabolome signatures associated with the DAP-R phenotype in S. mitis-oralis. In addition, these data provide support for further studying the use of strategic S. mitis-oralis metabolic inhibitors in additional strain-sets to resensitize DAP-R strains to DAP, using in vitro, ex vivo and in vivo models. Disclosures All authors: No reported disclosures.