GAPDH Activity Research Articles

Hexokinase 1 Cellular Localization Regulates the Metabolic Fate of Glucose

The product of hexokinase (HK) enzymes, glucose-6-phosphate, can be metabolized through glycolysis or directed to alternative pathways, such as the pentose phosphate pathway (PPP) to generate anabolic intermediates. 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. 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 glycolytic block at GAPDH, which is mediated by binding of cytosolic HK1 with S100A8/A9 and increased GAPDH nitrosylation through iNOS. Additionally, 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. Our data indicate that HK1 mitochondrial-binding alters glucose metabolism through regulation of GAPDH.

Determining the quantitative relationship between glycolysis and GAPDH in cancer cells exhibiting the Warburg effect

Previous studies have identified GAPDH as a promising target for treating cancer and modulating immunity because its inhibition reduces glycolysis in cells (cancer cells and immune cells) with the Warburg effect, a modified form of cellular metabolism found in cancer cells. However, the quantitative relationship between GAPDH and the aerobic glycolysis remains unknown. Here, using siRNA-mediated knockdown of GAPDH expression and iodoacetate-dependent inhibition of enzyme activity, we examined the quantitative relationship between GAPDH activity and glycolysis rate. We found that glycolytic rates were unaffected by the reduction of GAPDH activity down to 19% ± 4.8% relative to untreated controls. However, further reduction of GAPDH activity below this level caused proportional reductions in the glycolysis rate. GAPDH knockdown or inhibition also simultaneously increased the concentration of glyceraldehyde 3-phosphate (GA3P, the substrate of GAPDH). This increased GA3P concentration countered the effect of GAPDH knockdown or inhibition and stabilized the glycolysis rate by promoting GAPDH activity. Mechanistically, the intracellular GA3P concentration is controlled by the Gibbs free energy of the reactions upstream of GAPDH. The thermodynamic state of the reactions along the glycolysis pathway was only affected when GAPDH activity was reduced below 19% ± 4.8%. Doing so moved the reactions catalyzed by GAPDH + PGK1 (phosphoglycerate kinase 1, the enzyme immediate downstream of GAPDH) away from the near-equilibrium state, revealing an important biochemical basis to interpret the rate control of glycolysis by GAPDH. Collectively, we resolved the numerical relationship between GAPDH and glycolysis in cancer cells with the Warburg effect and interpreted the underlying mechanism.

140 Relaxin Regulates Porcine Adipose Tissue Development by Inhibiting Preadipocyte Number, Stimulating Lipolysis, and Upregulating mRNA Expression of Adipokine, Fatty Acid Metabolism and Extracellular Matrix Genes

Abstract Relaxin (RLN) is best known as a reproductive hormone that remodels cervical connective tissue. However, porcine adipose tissue can secrete and respond to RLN, suggesting locally derived RLN could modulate adipose tissue development and function. To test this hypothesis, primary cultures of porcine subcutaneous adipose tissue stromal-vascular (AT-SV) cells and visceral adipose tissue explants were utilized as model systems to determine the effect of 100 ng/ml exogenous RLN on 1) preadipocyte proliferation and differentiation, 2) the mRNA expression of adipokine, extracellular matrix protein and fatty acid metabolism genes, and 3) lipolytic rate in porcine adipose tissue. AT-SV cells were harvested from 3-day-old neonatal pigs (n=6) and plated in vitro in growth medium containing either vehicle or RLN for cell number assays or were induced to differentiate by treatment with insulin, hydrocortisone, and rosiglitazone for 8 days in the presence or absence of RLN for adipogenesis assays. Total RNA was extracted from differentiating cultures on days 0 and 8 post-induction. Real-time PCR was then utilized to determine changes in mRNA expression for target genes. For lipolysis and cell signaling experiments, visceral adipose tissue was obtained from market weight pigs at harvest, minced into explants, and cultured in serum-free medium in the presence or absence of RLN and cAMP pathway modulators with differences in glycerol release determined following 2-hour treatment. Overall, RLN decreased preadipocyte number 1.27-fold under serum-free conditions (P < 0.05), enhanced adipogenesis based upon 2.4-fold increase in GAPDH activity (P < 0.05) and a 2.2-fold increase in ORO accumulation (P < 0.05) while significantly increasing the mRNA expression of multiple metabolic, adipokine, and extracellular matrix component genes. RLN treatment stimulated glycerol release by 3-fold via the cAMP pathway (P < 0.01). Collectively, these data support the hypothesis that RLN regulates adipose tissue development through stimulating adipogenesis and modulating adipocyte metabolism.

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).

Antiplatelet Effect of Carbon Monoxide Is Mediated by NAD + and ATP Depletion

Carbon monoxide (CO) produced by haem oxygenases or released by CO-releasing molecules (CORM) affords antiplatelet effects, but the mechanism involved has not been defined. Here, we tested the hypothesis that CO-induced inhibition of human platelet aggregation is mediated by modulation of platelet bioenergetics. Approach and Results: To analyze the effects of CORM-A1 on human platelet aggregation and bioenergetics, a light transmission aggregometry, Seahorse XFe technique and liquid chromatography tandem-mass spectrometry-based metabolomics were used. CORM-A1-induced inhibition of platelet aggregation was accompanied by the inhibition of mitochondrial respiration and glycolysis. Interestingly, specific inhibitors of these processes applied individually, in contrast to combined treatment, did not inhibit platelet aggregation considerably. A CORM-A1-induced delay of tricarboxylic acid cycle was associated with oxidized nicotinamide adenine dinucleotide (NAD+) depletion, compatible with the inhibition of oxidative phosphorylation. CORM-A1 provoked an increase in concentrations of proximal (before GAPDH [glyceraldehyde 3-phosphate dehydrogenase]), but not distal glycolysis metabolites, suggesting that CO delayed glycolysis at the level of NAD+-dependent GAPDH; however, GAPDH activity was directly not inhibited. In the presence of exogenous pyruvate, CORM-A1-induced inhibition of platelet aggregation and glycolysis were lost, but were restored by the inhibition of lactate dehydrogenase, involved in cytosolic NAD+ regeneration, pointing out to the key role of NAD+ depletion in the inhibition of platelet bioenergetics by CORM-A1. The antiplatelet effect of CO is mediated by inhibition of mitochondrial respiration-attributed to the inhibition of cytochrome c oxidase, and inhibition of glycolysis-ascribed to cytosolic NAD+ depletion.

293-OR: Mitochondrial Hexokinase Dissociation Correlates with Obesity-Induced Inflammation and Regulates the Fate of Glucose

It has been shown that high fat diet (HFD) induced obesity triggers local immune cell infiltration leading to pro-inflammatory cytokine production and subsequently to a state of chronic low-grade inflammation in adipose and spleen tissues. However, the underlying mechanisms driving this chronic inflammation remains poorly defined. The product of hexokinase (HK) enzymes, glucose-6-phosphate, can be metabolized through glycolysis to produce ATP and pyruvate or alternative pathways, such as the pentose-phosphate-pathway (PPP) to generate anabolic intermediates. HK1 contains an N-terminal domain that permits mitochondrial binding, but its physiologic significance is unclear. We found that adipose and spleen tissues from HFD mice had increased cytosolic HK1, which correlated with an increase in inflammatory cytokine mRNA levels. To elucidate whether HK1 subcellular localization to the mitochondria affects the inflammatory response we generated mice lacking the HK1 mitochondrial binding domain, termed ΔE1HK1. These mice appear normal and show no change in body weight, Mendelian ratios, glucose tolerance or hexokinase activity across different tissues as compared to littermate controls. However, lipopolysaccharide challenge in these mice produced a hyper-inflammatory phenotype in vitro and in vivo. We performed steady-state and flux metabolomics in ΔE1HK1 mouse tissues and found increased glucose derived metabolites above the level of GAPDH, including PPP metabolites. Additionally, we found that GAPDH activity was attenuated in the ΔE1HK1 mice and may explain the elevated PPP flux upstream of GAPDH. Since PPP is vital for proper inflammatory cell activation, we treated cells from these mice with PPP inhibitors and found a reversal of the hyper-inflammatory phenotype observed in ΔE1HK1 mice. Our data suggests that mitochondrial dissociation of HK1 alters glucose utilization, which subsequently increases the pro-inflammatory response. Disclosure F. Keyhani Nejad: None. A. DeJesus: None. J.A. Geier: None. H. Ardehali: None.

Methylglyoxal couples metabolic and translational control of Notch signalling in mammalian neural stem cells

Gene regulation and metabolism are two fundamental processes that coordinate the self-renewal and differentiation of neural precursor cells (NPCs) in the developing mammalian brain. However, little is known about how metabolic signals instruct gene expression to control NPC homeostasis. Here, we show that methylglyoxal, a glycolytic intermediate metabolite, modulates Notch signalling to regulate NPC fate decision. We find that increased methylglyoxal suppresses the translation of Notch1 receptor mRNA in mouse and human NPCs, which is mediated by binding of the glycolytic enzyme GAPDH to an AU-rich region within Notch1 3ʹUTR. Interestingly, methylglyoxal inhibits the enzymatic activity of GAPDH and engages it as an RNA-binding protein to suppress Notch1 translation. Reducing GAPDH levels or restoring Notch signalling rescues methylglyoxal-induced NPC depletion and premature differentiation in the developing mouse cortex. Taken together, our data indicates that methylglyoxal couples the metabolic and translational control of Notch signalling to control NPC homeostasis.

The Highlighted Role of GAPDH and Nitric-Oxide Synthase Regulator Activity in Proton Beam Irradiated Melanoma BLM Cells.

Introduction: The human melanoma is a type of invasive tumor the treatment of which is challenging. To better understand the proton irradiation mechanisms as one of the widely applied therapy for this type of cancer, bioinformatics analysis of proteomics outcome could be beneficial. Methods: Protein-protein interaction network analysis of the differentially expressed proteins (DEPs) of melanoma BLM (BRO lung metastasis) cells in the treatment of 3 Gy dosage proton therapy was performed in this study via Cytoscape V.3.7.2. and its integrated plug-ins. Results: Eighteen DEPs were searched for network constructions and limited numbers of query +neighbor proteins were found central. The hub-bottlenecks (i.e. central nodes) were GAPDH, ACTB, ALB, AKT1, TP53, and EGFR. The fist mentioned proteins were from DEPs. The enrichment analysis of these elements identified nitric-oxide synthase regulator activity and the positive regulation of the norepinephrine uptake that may be the key to the mechanisms of proton therapy. Conclusion: In conclusion, the identified central nodes (EGFR, TP53, ALB, AKT1, GAPDH, and ACTB) and the related biological terms are the critical affected genes and biological terms in the irradiated melanoma cells.

Site-specific chemoproteomic profiling of targets of glyoxal.

Aim: Advanced glycation end products (AGE) are the biomarkers of aging and diabetes which are formed via reactions between glycating agents and biomacromolecules. However, no proteomic study has been reported to systematically investigate the protein substrates of AGEs. Results: In this paper, we used an aniline-based probe to capture the glyoxal-imine intermediate which is the transition sate of glyoxal-derived AGEs. Combined with the tandem orthogonal proteolysis activity-based protein profiling strategy, we successfully identified 962 lysines modified by glyoxal. Conclusion: Enzymes in glycolysis are heavily modified by glyoxal and our biochemical experiments showed that glyoxal can significantly inhibit the activity of GAPDH and glycolysis. These data indicated that AGEs modifications may contribute to pathological processes through impairing the glycolytic process.

Metabolic Alterations May Contribute to Cabozantinib Resistance in Acute Myeloid Leukemia Cells with FLT3-ITD

Background: Internal tandem duplication in the juxtamembranal region of FLT3 gene (FLT3-ITD) is one of the most common mutations in acute myeloid leukemia (AML), resulting in constitutive activation of FLT3 signaling pathway. Therefore FLT3 have been proved to be as a useful target for AML treatment. Previously, we demonstrated that cabozantinib, an oral multi-target tyrosine kinase inhibitor (TKI), could selectively cytotoxic to AML cells with FLT3-ITD (MV4-11 and Molm13). Recently, cabozantinib was reported to be well tolerated in AML patients with FLT3-ITD and potentially be useful in the treatment of AML with FLT3-ITD. However, it is known that TKI-resistance in AML often cause higher relapse rates and lower survival rates. In order to study the drug resistance mechanism, we established cabozantinib-resistant cell lines from MV4-11 and Molm13 cells, after gradual escalating concentration of cabozantinib incubation, with increasing IC50 from 9.5nM to 1.5μM and from 1.06nM to 473.36nM, respectively. The cabozantinib-resistance cell lines were named MV4-11 XR and Molm-13 XR, respectively.Aims: To elucidate the mechanism of survival advantage of cabozantinib-resistance of MV4-11-XR and Molm13-XR.Materials and Methods: The differential expression genes (DEGs) were examined using RNA-seq (Illumina NextSeq-500). Metascape recourse and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses were performed to predict the biological functions of DEGs. Quantitative PCR (Q-PCR) were used to validate the RNA-seq results. The extracellular acidification rate (ECAR) was measured using Seahorse bio-analyzer. In addition, to investigate the mitochondrial metabolism, analyses of oxygen consumption rate (OCR), glucose uptake, GAPDH activity, lactate production, ATP content were performed.Results: The gene expression profile in MV4-11 cells and Molm13 cells were used as baselines to establish the up- or down-regulated genes in MV4-11-XR and Molm13-XR cells, respectively. The FPKM were estimated with the selection criteria of q value<0.05 and [log2 (fold change0)>1 or <1 for significantly differential expression for up- and down-regulation, respectively. We identified a total of 1113 DEGs between the MV4-11 and MV4-11-XR cells, and a total of 1057 DEGs between the Molm13 and Molm13-XR cells. By using KEGG Mapper to interrogate pathways significance, we found that the metabolic pathway was the most significant in both up- and down-regulated DEGs in both resistant cells (MV4-11-XR up: 30 DEGs, down: 52 DEGs; Molm13-XR up: 27 DEGs, down: 58 DEGs). The information suggests that metabolic alterations occur in both drug resistant cell lines. Both MV4-11-XR and Molm13-XR cells showed higher glucose uptake, GAPDH activity, lactate production and ATP content than corresponding parental cells. Consistent with increased lactate export, analysis of glycolytic function showed a significant increase in glycolysis, glycolytic capacity and glycolytic reserve in both resistant cells. In addition, we showed increased basal mitochondrial and ATP-coupled respiration in both resistant cells, compared to their corresponding parental cells. Finally, decreased OCR / ECAR ratios indicated the relatively higher reliance on glycolysis in both resistant cells: 15.79 in Molm13-XR cells compared to 24.93 in Molm-13 cells, and 1.75 in MV4-11-XR cells compared to 9.97 in MV4-11 cells.Conclusion: Our study highlights that alteration of metabolic pathways may contribute cabozantinib resistance. We suggest that targeting these pathways may be a viable strategy to overcome cabozantinib resistance. DisclosuresNo relevant conflicts of interest to declare.

PO-087 Oxidative stress as a selective anticancer agent: preclinical evaluation of a targeted combination strategy for mutant P53 non-small cell lung cancer

IntroductionOsteosarcoma (OS) is the most common primary bone sarcoma that mainly occurs in children and adolescents. The existence of drug resistant cancer stem cells (CSCs) with progenitor properties is responsible for OS relapse and metastasis. Thus, development of specific therapies targeting OS-CSCs is necessary to increase the long-term survival rate. Although ascorbic acid (AA) has controversial history as anticancer agent, recently it has been re-evaluated revealing more cytotoxic effect to cancer than normal cells. The aim of the study was to analyse AA as potential therapeutic for selective targeting of OS-CSCs.Material and methodsTo establish primary tumour cultures, tumour samples were mechanically dissected and enzymatically digested. Sarcosphere assay was used to isolate OS-CSCs. The cytotoxic effect of AA was determined by MTT assay as well as relationship between cell concentration and AA. OS-CSCs were treated with different concentrations of AA (2.5–55 µg/ml) during 72 hour. Concentrations of AA used for further experiments were 30 µg/ml and 40 µg/ml, respectively. Effect of AA on sarcosphere-forming ability was measured under low-attachment condition during 28 days. Cell death type was determined by Annexin V/PI staining using flow cytometry. Levels of GAPDH were determined by western blot while ROS were measured by DCFH-DA assay. Seahorse XF analyzer was used to measure glycolysis and oxidative phosphorylation.Results and discussionsWhile AA did not have any effect on hMSCs, U2OS and Hek 293, respectively, AA efficiently induced dose-dependent viability reduction of OS-CSCs. Further, it can be concluded that IC50 values of AA depend on the number of seeded OS-CSCs. AA successfully reduced sarcosphere formation on 6th day. High cytotoxicity of AA was further confirmed by Annexin V/PI staining. Prevalent death mode induced by AA was apoptotic since more than 70% of Annexin V-positive cells were detected. In addition, AA inhibited the activity of the key glycolytic enzyme GAPDH and induced ROS levels. Following the treatment with AA, extracellular acidification rate as a measure of glycolysis, was reduced significantly. Moreover, AA increased metabolic potential of OS-CSCs implying cells’ ability to meet an energy demand via respiration and glycolysis.ConclusionBased on the obtained results, it can be concluded that AA selectively targets OS-CSCs. The death mechanism is based on the blockage of glycolytic cycle and increased intracellular levels of ROS.

Cadmium inhibits lysine acetylation and succinylation inducing testicular injury of mouse during development

The toxic effects of cadmium (Cd) in the reproductive system have been confirmed, and lysine acetylation and succinylation play important roles in spermatogenesis. However, little attention determined whether Cd could affect lysine acylation and how it might have an impact on the reproductive system. Therefore, with the goal of contributing to this subject, we have examined the effects of Cd on lysine acetylation and succinylation of proteins in the germ cells of male mice testes during different developmental stages. We adopted intraperitoneal injection of cadmium chloride (1.2 mg/kg body weight) in mice once every 5 days from postnatal day 5–60. The results showed that Cd could restrict GAPDH activity, ATP and cAMP levels of germ cells to inhibit lysine acetylation and succinylation in the testes, inducing reproductive injuries. Cd also restricts acetylation of histone H4K5 and H4K12, which could result in failure of spermiogenesis. Remarkably, polarized acetylation occurs in meiosis, and high-level acetylation occurs earlier than high-level succinylation during spermatogenesis. Moreover, Cd has a limited effect on body weight but reduces the weight of the testis and litter size. Our research may provide a new way to reveal the mechanisms of Cd reproductive toxicity related to lysine acetylation and succinylation.

The role of resveratrol and melatonin in the nitric oxide and its oxidation products mediated functional and structural modifications of two glycolytic enzymes: GAPDH and LDH

BackgroundNitric oxide is a well-known gaseous signaling molecule and protein modifying agent. However, at higher concentrations or during oxidative stress nitric oxide may exert some deleterious effects on protein structure and function. Here we investigated the influence of nitric oxide and products of its oxidation on two glycolytic enzymes: GAPDH and LDH under in vitro nitrosative stress conditions. Secondly, we applied natural antioxidants: melatonin and resveratrol to examine their effects on the enzymes under studied conditions. MethodsBy means of UV–VIS and fluorescence spectroscopy methods we compared nitric oxide mediated changes of enzyme activities, amount of free sulfhydryl groups (-SH) and bis-ANS probe binding. Finally, we predicted potential cysteine residues modified by nitric oxide in studied proteins using GPS-SNO software. ResultsOur results indicated that nitric oxide reversibly inactivates GAPDH but does not affect the activity of LDH. Nitric oxide dependent GAPDH activity decline was accompanied by the reduction of the amount of free –SH groups and GAPDH-bound bis-ANS fluorescence. Reduction of the number of free –SH groups and protein-bound bis-ANS fluorescence was also observed in LDH treated with NO. Applied antioxidants increased inactivation of GAPDH and structural changes of GAPDH and LDH. ConclusionsNitric oxide modifies function and structure of thiol-dependent enzyme such as GAPDH and structure of LDH which function do not rely on cysteine thiols. Both resveratrol and melatonin exerted prooxidative properties in studied conditions. General significanceExtensively studied antioxidants: resveratrol and melatonin may function as a prooxidative species under in vitro nitrosative stress conditions.

Differential killing and radio-modifying effects of iodoacetate in mammalian normal and cancer cells.

To explore possible applications of iodoacetate (IA), a glycolytic inhibitor, in cancer treatment, we screened its cytotoxicity and radioprotective/sensitizing efficacy in three different mammalian cell lines; A549 (human lung carcinoma), MCF7 (human mammary cancer), a non-cancerous CHO (Chinese hamster ovary) cells and human lymphocytes. Experiments were carried out using IA concentrations ranging from 0.01 to 2.5µg/ml, with or without 60Coγ-radiation. In the outcomes, IA was found to exhibit higher toxicity in the cancer cells, whereas it was non-toxic/marginally toxic to the non-cancerous cells. Considerably higher glucose uptake in both cancer cells lines was observed indicating higher rates of glycolysis. IA significantly inhibited glycolysis as reflected by GAPDH activity inhibition. Radiomodifying effects of IA were found to be concentration dependent in both cancerous and non-cancerous cells. The response in non-cancerous was found to be biphasic: at lower concentrations, it offered significant radioprotection; however, the protection decreased with increasing concentration. Moreover, at the highest tested concentration, marginal radiosensitization was also observed (as indicated by clonogenic assay). In both cancer cells, IA offered significant amount of radiosensitization which was considerably high at higher concentrations. Further experiments were carried out to estimate the Dose Modification Factor (DMF) to quantify and compare relative radiosensitization by IA in cancer and normal cell lines. The DMF was calculated for three different concentrations of IA, 0.5, 1, and 1.5µg/ml, and corresponding values were found to be 1.26, 1.43, and 1.89 for A549 cancer cells, whereas for normal CHO cells, it was 1.13, 1.13, and 1.24. In conclusion, differential killing and radiosensitizing effects of IA suggest that it may have potential use as a anticancer agent and radiosensitizer in cancer therapy.

The NLRP3 inflammasome contributes to sarcopenia and lower muscle glycolytic potential in old mice.

The mechanisms underpinning decreased skeletal muscle strength and slowing of movement during aging are ill-defined. "Inflammaging," increased inflammation with advancing age, may contribute to aspects of sarcopenia, but little is known about the participatory immune components. We discovered that aging was associated with increased caspase-1 activity in mouse skeletal muscle. We hypothesized that the caspase-1-containing NLRP3 inflammasome contributes to sarcopenia in mice. Male C57BL/6J wild-type (WT) and NLRP3-/- mice were aged to 10 (adult) and 24 mo (old). NLRP3-/- mice were protected from decreased muscle mass (relative to body mass) and decreased size of type IIB and IIA myofibers, which occurred between 10 and 24 mo of age in WT mice. Old NLRP3-/- mice also had increased relative muscle strength and endurance and were protected from age-related increases in the number of myopathic fibers. We found no evidence of age-related or NLRP3-dependent changes in markers of systemic inflammation. Increased caspase-1 activity was associated with GAPDH proteolysis and reduced GAPDH enzymatic activity in skeletal muscles from old WT mice. Aging did not alter caspase-1 activity, GAPDH proteolysis, or GAPDH activity in skeletal muscles of NLRP3-/- mice. Our results show that the NLRP3 inflammasome participates in age-related loss of muscle glycolytic potential. Deletion of NLRP3 mitigates both the decline in glycolytic myofiber size and the reduced activity of glycolytic enzymes in muscle during aging. We propose that the etiology of sarcopenia involves direct communication between immune responses and metabolic flux in skeletal muscle.

Transcriptional deregulation of genetic biomarkers in Chironomus riparius larvae exposed to ecologically relevant concentrations of di(2-ethylhexyl) phthalate (DEHP).

Di(2-ethylhexyl) phthalate (DEHP) is a ubiquitous environmental pollutant used worldwide as a plasticizer and solvent in many formulations. Based on available toxicological data, it has been classified as toxic for reproduction and as an endocrine disruptor. Despite this, ecotoxicological studies in aquatic wildlife organisms are still scarce. In the present work, the toxic molecular alterations caused by DEHP in aquatic larvae of the midge Chironomus riparius have been studied, by analyzing the transcriptional activity of genes related to some vital cellular pathways, such as the ribosomal machinery (rpL4, rpL13), the cell stress response (hsc70, hsp70, hsp40, hsp27), the ecdysone hormone pathway (EcR), the energy metabolism (GAPDH), and detoxication processes (CYP4G). Environmentally relevant concentrations (10−3 to 105 μg/L) and exposure conditions (24 to 96 h) have been tested, as well as the toxic effects after DEHP withdrawal. Although the compound caused no mortality, significant changes were detected in almost all the studied biomarkers: e.g. strong repression of hsp70; general inhibition of EcR; GAPDH activity loss in long exposures; among others. Our data show a general transcriptional downregulation that could be associated with an adaptive response to cell damage. Besides, the activity of the compound as an ecdysone antagonist and its delayed effects over almost all the biomarkers analyzed are described as novel toxic targets in insects.

Effect of GABRA1 Cytoplasmic Peptide on a Regulatory Protein

The pentameric neurotransmitter-gated chloride channel, GABA (type A) receptor, is well-recognized for phasic and tonic neuronal inhibition. The phasic response at the synapse is due to receptors typically containing two alpha, two beta, and one gamma subunit. Half of all GABA (type A) receptors in adult human brain have the alpha (type 1), or alpha-1, subunit (gene ID: GABRA1). Each consists of an extracellular ligand-binding domain and four transmembrane helices (TM1-4), both of which are highly conserved, as well as a large intracellular loop domain (ILD), whose structure is unknown. The human receptor is crystallized without the ILD, which connects TM3 and TM4, that is engineered out. The topology and function of the 86-residue alpha-1 ILD remains to be fully elucidated. GAPDH (d-glyceraldehyde-3-phosphate dehydrogenase) phosphorylates specific ILD residues, regulating receptor function. In this study, we examined the effect of a synthesized ILD peptide (residues 335 to 364 from GABRA1) on the oxidoreductase activity of GAPDH. The custom peptide, containing the first 30 cytosolic residues, was pre-incubated with GAPDH, and enzyme activity measured by NADH absorbance (340nm) upon oxidation of the substrate d-glyceraldehyde. When pre-incubated for 1hr at 20degC, the ILD peptide inhibited enzyme activity in a concentration-dependent manner showing an exponential non-linear function (y=a∗e ˆ (b/x+c)). The half-maximum inhibition occurred at a [peptide] = 8.7micromolar. When pre-incubated for 48hr at 4degC, the ILD peptide further enhanced inhibition of oxidoreductase activity, suggesting that hydrophobic forces play a role in the interaction between the ILD peptide and GAPDH. Molecular modeling experiments indicate that the ILD binding site on GAPDH is in close proximity to the phosphatidylserine-binding site on GAPDH. The findings may contribute to our understanding of receptor function and point to possible treatments in conditions, such as Alzheimer's dementia, where GABA receptor pharmacology is shown to play a role.

Crystal Structures of Group B Streptococcus Glyceraldehyde-3-Phosphate Dehydrogenase: Apo-Form, Binary and Ternary Complexes.

Glyceraldehyde 3-phosphate dehydrogenase or GAPDH is an evolutionarily conserved glycolytic enzyme. It catalyzes the two step oxidative phosphorylation of D-glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate using inorganic phosphate and NAD+ as cofactor. GAPDH of Group B Streptococcus is a major virulence factor and a potential vaccine candidate. Moreover, since GAPDH activity is essential for bacterial growth it may serve as a possible drug target. Crystal structures of Group B Streptococcus GAPDH in the apo-form, two different binary complexes and the ternary complex are described here. The two binary complexes contained NAD+ bound to 2 (mixed-holo) or 4 (holo) subunits of the tetrameric protein. The structure of the mixed-holo complex reveals the effects of NAD+ binding on the conformation of the protein. In the ternary complex, the phosphate group of the substrate was bound to the new Pi site in all four subunits. Comparison with the structure of human GAPDH showed several differences near the adenosyl binding pocket in Group B Streptococcus GAPDH. The structures also reveal at least three surface-exposed areas that differ in amino acid sequence compared to the corresponding areas of human GAPDH.

Abstract 4968: “Moonlighting Functions” of glycolytic enzymes relate to human prostate cancer invasion

Abstract Background: Castrate-resistant prostate cancer (CRPC) is the second leading cause of cancer deaths among US men. Currently, a large number of small and low grade prostate cancers (PCa) are being diagnosed. Only a few of them will metastasize and become lethal. A clinical method to distinguish aggressive from the indolent tumors is warranted. An enhanced glucose metabolism (Warbürg effect) and invasion of cells from their organs of origin and metastasis to distant organs are two characteristics that are ubiquitous in most solid tumors. In the last few years, the non-glycolytic activities (“moonlighting functions”) of glycolytic enzymes have been investigated in relation to cancer cell invasion. We propose that oxidatively stressed PCa cells, as a countermeasure, redirect some of the glycolytic enzymes to their moonlighting functions. Methods: We used a novel microfluidic device that separates the invading from the non-invading PCa cells based on their migratory characteristics in a 3D collagen matrix. We performed ICC analysis of the separated cells for oxidative stress generating enzyme spermidine/spermine N1 acetyl transferase (SSAT) and glycolytic enzymes aldolase, GAPDH and F-actin levels. We also carried out proteomic analysis of aldolase and GAPDH levels and oxidation state and metabolomic analysis for enzymatic activities of those two enzymes. Results: Under identical condition, LNCaP cells show minimum invasion, whereas between 30-40% of C4-2 cells invade more than 0.8 mm in 16 hours. ICC assay of C4-2 cells shows that most migratory C4-2 cells have SSAT overexpression, but less than 5% stationary cells show this effect. Proteomic analysis shows that androgen treatment that increases overall oxidative stress in both LNCaP and C4-2 cells actually decreases oxidation status of GAPDH in LNCaP cells and increases that in C4-2 cells. Anti-androgen enzalutamide reverses the androgen effect in both cell lines. The glycolytic activity of GAPDH decreases with an increase in protein oxidation. Our metabolomic data further confirmed an increase in GAPDH activity in LNCaP and a decrease in C4-2 cells after androgen treatment and the reversal with enzalutamide as expected from its oxidation status. Discussion: SSAT expression and a consequent increase in oxidative stress are related to invasion of CRPC. Oxidation of certain glycolytic enzymes in CRPC cells may divert them to their moonlighting function related to cellular invasion and metastasis. These functions may be monitored in patient biopsies and/or prostatectomy tissues for PCa prognosis. Application of the microfluidic method for the separation of invading from the non-invading cells and proteogenomic and metabolomic analysis of these cells isolated from patient prostatectomy tissues are currently being standardized. Citation Format: Jiaquan Yu, Ashley M. Weichmann, Alexandria Craig, Wei Huang, Dawn R. Church, Farideh Mehraein, Laurie L. Parker, David J. Beebe, George Wilding, Hirak S. Basu. “Moonlighting Functions” of glycolytic enzymes relate to human prostate cancer invasion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4968.

Memory CD8+ T Cells Require Increased Concentrations of Acetate Induced by Stress for Optimal Function

How systemic metabolic alterations during acute infections impact immune cell function remains poorly understood. We found that acetate accumulates in the serum within hours of systemic bacterial infections and that these increased acetate concentrations are required for optimal memory CD8(+) Tcell function invitro and invivo. Mechanistically, upon uptake by memory CD8(+) Tcells, stress levels of acetate expanded the cellular acetyl-coenzyme A pool via ATP citrate lyase and promoted acetylation of the enzyme GAPDH. This context-dependent post-translational modification enhanced GAPDH activity, catalyzing glycolysis and thus boosting rapid memory CD8(+) Tcell responses. Accordingly, in a murine Listeria monocytogenes model, transfer of acetate-augmented memory CD8(+) Tcells exerted superior immune control compared to control cells. Our results demonstrate that increased systemic acetate concentrations are functionally integrated by CD8(+) Tcells and translate into increased glycolytic andfunctional capacity. The immune system thus directly relates systemic metabolism with immune alertness.